Product and process for transformation of thraustochytriales microorganisms

ABSTRACT

Disclosed are nucleic acid and amino acid sequences for acetolactate synthase, acetolactate synthase regulatory regions, α-tubulin promoter, a promoter from a Thraustochytriales polyketide synthase (PKS) system, and fatty acid desaturase promoter, each from a Thraustochytriales microorganism. Also disclosed are recombinant vectors useful for transformation of Thraustochytriales microorganisms, as well as a method of transformation of Thraustochytriales microorganisms. The recombinant nucleic acid molecules of the present invention can be used for the expression of foreign nucleic acids in a Thraustochytriales microorganism as well as for the deletion, mutation, or inactivation of genes in Thraustochytriales microorganisms.

CROSS-REFERENCE TO RELATED APPLICATIONS

This application is a continuation of U.S. application Ser. No.12/942,759, filed Nov. 9, 2010, which is a continuation of U.S.application Ser. No. 11/925,885, filed Oct. 27, 2007, now U.S. Pat. No.7,851,191, which is a continuation of U.S. application Ser. No.11/021,712, filed Dec. 22, 2004, now U.S. Pat. No. 8,206,984, which is acontinuation of U.S. application Ser. No. 10/124,807, filed Apr. 16,2002, now U.S. Pat. No. 7,001,772, which claims the benefit of priorityunder 35 U.S.C. §119(e) to U.S. Provisional Application Ser. No.60/284,116, filed Apr. 16, 2001, entitled “Product and Process forTransformation of Thraustochytriales Microorganisms”. The entiredisclosure of U.S. Provisional Application Ser. No. 60/284,116 and U.S.application Ser. Nos. 10/124,807, 11/021,712, 11/925,885 and 12/942,759are hereby incorporated herein by reference.

FIELD OF THE INVENTION

This invention generally relates to an isolated nucleic acid moleculeencoding a Thraustochytriales acetolactate synthase, includingacetolactate synthases that confer reduced sensitivity to sulfonylureacompounds, imidazolinone-class inhibitors and/or pyrimidinyloxybenzoates, onto microorganisms of the order Thraustochytriales; torecombinant nucleic acid molecules comprising selectable markers usefulfor the transformation of microorganisms of the orderThraustochytriales, and to methods of transforming such microorganismsusing recombinant nucleic acid molecules of the present invention. Thepresent invention also relates to gene promoters useful inThraustochytriales expression systems. The recombinant nucleic acidmolecules of the present invention can be used for the expression offoreign nucleic acids in a Thraustochytriales microorganism as well asfor the deletion, mutation, or inactivation of genes inThraustochytriales microorganisms.

BACKGROUND OF THE INVENTION

Developments have resulted in revision of the taxonomy of theThraustochytrids. Taxonomic theorists place Thraustochytrids with thealgae or algae-like protists. However, because of taxonomic uncertainty,it would be best for the purposes of the present invention to considerthe strains described in the present invention as Thraustochytrids(Order: Thraustochytriales; Family: Thraustochytriaceae; Genus:Thraustochytrium, Schizochytrium, Labyrinthuloides, or Japonochytrium).Taxonomic changes are summarized below.

Strains of certain unicellular microorganisms disclosed and claimedherein are members of the order Thraustochytriales. Thraustochytrids aremarine eukaryotes with a problematic taxonomic history. Problems withthe taxonomic placement of the Thraustochytrids have been reviewed byMoss (1986), Bahnweb and Jackie (1986) and Chamberlain and Moss (1988).

For convenience purposes, the Thraustochytrids were first placed bytaxonomists with other colorless zoosporic eukaryotes in thePhycomycetes (algae-like fungi). The name Phycomycetes, however, waseventually dropped from taxonomic status, and the Thraustochytrids wereretained in the Oomycetes (the biflagellate zoosporic fungi). It wasinitially assumed that the Oomycetes were related to the heterokontalgae, and eventually a wide range of ultrastructural and biochemicalstudies, summarized by Barr (1983) supported this assumption. TheOomycetes were in fact accepted by Leedale (1974) and other phycologistsas part of the heterokont algae. However, as a matter of convenienceresulting from their heterotrophic nature, the Oomycetes andThraustochytrids have been largely studied by mycologists (scientistswho study fungi) rather than phycologists (scientists who study algae).

From another taxonomic perspective, evolutionary biologists havedeveloped two general schools of thought as to how eukaryotes evolved.One theory proposes an exogenous origin of membrane-bound organellesthrough a series of endosymbioses (Margulis 1970); e.g., mitochondriawere derived from bacterial endosymbionts, chloroplasts fromcyanophytes, and flagella from spirochaetes. The other theory suggests agradual evolution of the membrane-bound organelles from thenon-membrane-bounded systems of the prokaryote ancestor via anautogenous process (Cavalier-Smith 1975). Both groups of evolutionarybiologists however, have removed the Oomycetes and Thraustochytrids fromthe fungi and place them either with the chromophyte algae in thekingdom Chromophyta (Cavalier-Smith 1981) (this kingdom has been morerecently expanded to include other protists and members of this kingdomare now called Stramenopiles) or with all algae in the kingdomProtoctista (Margulis and Sagan (1985).

With the development of electron microscopy, studies on theultrastructure of the zoospores of two genera of Thraustochytrids,Thraustochytrium and Schizochytrium, (Perkins 1976; Kazama 1980; Barr1981) have provided good evidence that the Thraustochytriaceae are onlydistantly related to the Oomycetes. Additionally, genetic datarepresenting a correspondence analysis (a form of multivariatestatistics) of 5 S ribosomal RNA sequences indicate thatThraustochytriales are clearly a unique group of eukaryotes, completelyseparate from the fungi, and most closely related to the red and brownalgae, and to members of the Oomycetes (Mannella et al. 1987). Mosttaxonomists have agreed to remove the Thraustochytrids from theOomycetes (Bartnicki-Garcia 1988).

In summary, employing the taxonomic system of Cavalier-Smith (1981,1983), the Thraustochytrids are classified with the chromophyte algae inthe kingdom Chromophyta, (Stramenopiles). This places them in acompletely different kingdom from the fungi, which are all placed in thekingdom Eufungi. The taxonomic placement of the Thraustochytrids istherefore summarized below:

Kingdom: Chromophyta (Stramenopiles)

Phylum: Heterokonta

Order: Thraustochytriales

Family: Thraustochytriaceae

Genus: Thraustochytrium, Schizochytrium, Labyrinthuloides, orJaponochytrium

Some early taxonomists separated a few original members of the genusThraustochytrium (those with an amoeboid life stage) into a separategenus called Ulkenia. However it is now known that most, if not all,Thraustochytrids (including Thraustochytrium and Schizochytrium),exhibit amoeboid stages and as such, Ulkenia is not considered by someto be a valid genus. As used herein, the genus Thraustochytrium willinclude Ulkenia.

Despite the uncertainty of taxonomic placement within higherclassifications of Phylum and Kingdom, the Thraustochytrids remain adistinctive and characteristic grouping whose members remainclassifiable within the order Thraustochytriales.

Schizochytrium and other Thraustochytriales microorganisms havesubstantial existing and potential commercial value because of theirability to produce large quantities of lipoidal compounds, includinghighly unsaturated fatty acids (HUFAs) and various carotenoids (e.g.,astaxanthin). Omega-3 highly unsaturated fatty acids are of significantcommercial interest in that they have been recently recognized asimportant dietary compounds for preventing arteriosclerosis and coronaryheart disease, for alleviating inflammatory conditions and for retardingthe growth of tumor cells. These beneficial effects are a result both ofomega-3 HUFAs causing competitive inhibition of compounds produced fromomega-6 fatty acids, and from beneficial compounds produced directlyfrom the omega-3 HUFAs themselves (Simopoulos et al., 1986). Omega-6fatty acids are the predominant HUFAs found in plants and animals.Therefore, further development of Thraustochytriales microorganisms ascommercial production organisms will benefit significantly from theability to make specific genetic changes to the organisms viarecombinant DNA technology, including enhancing the production of thehighly valuable HUFAs and carotenoids by such organisms. In addition,the ability to gain a better understanding of the biochemistry andmolecular biology of this poorly characterized group of organisms willprovide valuable information that can be used to guide future straindevelopment efforts. Prior to the present invention, however, methodsand recombinant constructs suitable for transforming Thraustochytrids,including members of the genera, Schizochytrium and Thraustochytriumwere not available. Importantly, the development of selectable markersthat are particularly useful for transforming Thraustochytrialesmicroorganisms and the identification of Thraustochytriales-specificpromoter sequences were not available prior to the present invention.

Prior investigators have described transformation methods and reagentsfor use in various microorganisms, including in microalgae that are notmembers of the Thraustochytriales order. U.S. Pat. No. 6,027,900 toAllnutt et al. discloses genetic fusions for use in genetic engineeringof eukaryotic algae, and particularly, Phaeodactylum tricornutum, usinga promoter for a photosynthetic algal light harvesting gene and the Shble gene from Streptoalloteichus hindustanus as a selectable marker. Thecells are grown in high concentrations of salt (e.g., 10-35 g/L) andZeocin™ for selection of transformants. The microalgal cells suitablefor transformation using such a method are photosynthetic microalgaethat can be grown under the high salt conditions. U.S. Pat. No.5,661,017 to Dunahay et al. discloses a method to transform cholorophyllC-containing algae (e.g., Diatoms) using a recombinant constructcomprising a selectable marker operatively linked to a regulatorycontrol sequence suitable for expression of the marker in thecholorophyll C-containing algae. The selectable marker is disclosed asbeing any suitable marker, including markers isolated from bacterial andfungal sources, and is preferably neomycin phosphotransferase. Theregulatory control sequence can include any regulatory sequence derivedfrom a cholorophyll C-containing algae, and preferably, from Cyclotellacryptica (e.g., a C. cryptica acetyl-CoA carboxylase regulatorysequence).

However, such methods are not readily transferable to the transformationof Thraustochytriales microorganisms, because, prior to the presentinvention, the transformation of microorganisms such asThraustochytriales (e.g., microalgae) was far from routine. Markers andtransformation systems that have become well developed for bacteria andyeast are not necessarily readily adaptable to other microorganisms.Indeed, U.S. Pat. No. 5,661,017 notes that “there has been littlesuccess in developing transformation systems for eucaryotic microalgae”(col. 1, lines 49-51), which is partly due to the difficulty ofintroducing foreign DNA into such microorganisms, and partly due to alack of suitable markers and vectors for use in such transformation. Thesystem described in U.S. Pat. No. 5,661,017 was developed specificallyfor the chlorophyll C-containing algae because those inventors believedthem to be amenable to genetic transformation, particularly as comparedto other algae. Similarly, U.S. Pat. No. 6,027,900, which teaches atransformation method that is specific to photosynthetic microalgae,speaks to the belief that most algae are refractory to any type ofgenetic manipulation (col. 1, lines 39-47). The systems adapted forbacteria, yeast, insect and animal cells have not been readily adaptedto microalgae. Therefore, prior to the present invention, there wasstill a need in the art for effective transformation systems and methodsthat are specific for microalgae.

Additionally, although the order Thraustochytriales is now grouped withthe chromophyte algae in the Stramenopiles, there is still an opinion bysome in the art that these microorganisms are quite different from mostmicroalgae, and some of those of skilled in the art have the opinionthat Thraustochytriales members may not be properly classified asmicroalgae at all. Microorganisms considered to be microalgae haveevolved at least four separate times during evolution, leading the“macroalgal” type microorganisms to be placed in different kingdoms(e.g. the red algae, green algae and golden algae (Chromophyta) are allin separate kingdoms). As a result, transformation systems that havebeen demonstrated to be useful in other microalgae are not expected tobe useful for Thraustochytriales. Therefore, despite the commercialvalue of Thraustochytriales microorganisms, the ability to make use ofthe full potential of such microorganisms by genetic engineering has notheretofore been realized. Prior to the present invention, the presentinventors were not aware of any promoters, selectable markers, orvectors useful for transformation of Thraustochytriales microorganisms,nor was there any knowledge regarding what selection systems could beused in or adapted to Thraustochytriales.

In summary, there is a need in the art to develop methods fortransforming Thraustochytriales microorganisms, thereby providing ameans to create strains with enhanced commercial value. In addition,there is a need in the art to develop methods for mutation orinactivation of specific genes by homologous or nonhomologousrecombination in Thraustochytriales microorganisms, providing a new wayto alter cellular metabolism and to identify the functions of specificgenes in Thraustochytriales.

SUMMARY OF THE INVENTION

One embodiment of the present invention relates to an isolated nucleicacid molecule comprising a nucleic acid sequence selected from the groupconsisting of: (a) a nucleic acid sequence encoding a protein having anamino acid sequence selected from the group consisting of SEQ ID NO:15,SEQ ID NO:19, SEQ ID NO:22 and SEQ ID NO:24, wherein the protein is anacetolactate synthase; (b) a nucleic acid sequence encoding a proteinhaving an amino acid sequence that is at least about 75% identical to anamino acid sequence of (a), wherein the protein is an acetolactatesynthase; and, (c) a nucleic acid sequence that is fully complementaryto the nucleic acid sequence of (a) or (b). In one aspect, such anucleic acid sequence encodes a protein having an amino acid sequencethat is at least about 85% identical to an amino acid sequence of (a),and wherein the protein is an acetolactate synthase. In another aspect,such a nucleic acid sequence encodes a protein having an amino acidsequence that is at least about 95% identical to an amino acid sequenceof (a), and wherein the protein is an acetolactate synthase. In yetanother aspect, such a nucleic acid sequence encodes a protein having anamino acid sequence that differs from SEQ ID NO:15 by an amino aciddeletion, insertion, or substitution at an amino acid position selectedfrom the group consisting of: 116G, 117A, 192P, 200A, 251K, 358M, 383D,592V, 595W, and 599F. In one aspect, the nucleic acid sequence encodes aprotein having an amino acid sequence selected from the group consistingof SEQ ID NO:15, SEQ ID NO:19, SEQ ID NO:22 and SEQ ID NO:24, andwherein the protein is an acetolactate synthase. In yet another aspect,the nucleic acid sequence is selected from the group consisting ofnucleotides 1260-3314 of SEQ ID NO:14, nucleotides 1260-3314 of SEQ IDNO:18, nucleotides 1260-3314 of SEQ ID NO:21, and nucleotides 1260-3314of SEQ ID NO:23.

Preferably, expression of the protein encoded by the nucleic acidsequences identified above confers reduced sensitivity to compoundsselected from the group consisting of: sulfonylurea compounds,imidazolinone-class inhibitors, and pyrimidinyl oxybenzoates, onto amicroorganism of the Order Thraustochytriales that is transformed withsuch a nucleic acid molecule. In one aspect of this embodiment, thenucleic acid sequence encodes a protein having an amino acid sequenceselected from the group consisting of SEQ ID NO:19, SEQ ID NO:22 and SEQID NO:24. In another aspect of this embodiment, the nucleic acidsequence is selected from the group consisting of: nucleotides 1260-3314of SEQ ID NO:14, nucleotides 1260-3314 of SEQ ID NO:18, nucleotides1260-3314 of SEQ ID NO:21, and nucleotides 1260-3314 of SEQ ID NO:23.

In one embodiment of the present invention, the nucleic acid sequencedescribed above encodes a Schizochytrium acetolactate synthase. In oneaspect, expression of the Schizochytrium acetolactate synthase confersreduced sensitivity to compounds selected from the group consisting of:sulfonylurea compounds, imidazolinone-class inhibitors, and pyrimidinyloxybenzoates, onto a microorganism of the Order Thraustochytriales thatis transformed with the nucleic acid molecule.

Another embodiment of the present invention relates to a recombinantnucleic acid molecule comprising any of the isolated nucleic acidmolecules described above, operatively linked to a transcription controlsequence. Another embodiment of the present invention relates to arecombinant microorganism of the order Thraustochytriales that istransformed with such a recombinant nucleic acid molecule.

Yet another embodiment of the present invention relates to a recombinantvector for transformation of microorganisms of the OrderThraustochytriales. The vector includes a nucleic acid sequence encodingan acetolactate synthase that confers reduced sensitivity to compoundsselected from the group consisting of: sulfonylurea compounds,imidazolinone-class inhibitors, and pyrimidinyl oxybenzoates, onto amicroorganism of the order Thraustochytriales. The acetolactate synthasehas an amino acid sequence selected from the group consisting of: (a) anamino acid sequence selected from the group consisting of SEQ ID NO:19,SEQ ID NO:22 and SEQ ID NO:24; and, (b) an amino acid sequence that isat least about 75% identical to an amino acid sequence of (a). Thenucleic acid sequence encoding an acetolactate synthase is operativelylinked to a transcription control sequence. In one aspect, therecombinant vector is an expression vector. In another aspect, therecombinant vector is a targeting vector. In other aspects, the nucleicacid sequence in the vector encodes an acetolactate synthase having anamino acid sequence that is at least about 85% identical, and in anotheraspect, at least about 95% identical, to an amino acid sequence of (a).In one aspect, the nucleic acid sequence encodes a protein having anamino acid sequence that differs from SEQ ID NO:15 by an amino aciddeletion, insertion, or substitution at an amino acid position selectedfrom the group consisting of: 116G, 117A, 192P, 200A, 251K, 358M, 383D,592V, 595W, and 599F. In a preferred aspect, the acetolactate synthasehas an amino acid sequence selected from the group consisting of SEQ IDNO:19, SEQ ID NO:22 and SEQ ID NO:24. In another aspect, the nucleicacid sequence is selected from the group consisting of: nucleotides1260-3314 of SEQ ID NO:18, nucleotides 1260-3314 of SEQ ID NO:21, andnucleotides 1260-3314 of SEQ ID NO:23. The transcription controlsequence in the recombinant vector can include, but is not limited to, aThraustochytriales α-tubulin promoter, a Thraustochytriales acetolactatesynthase promoter, a promoter from a Thraustochytriales polyketidesynthase (PKS) system, or a Thraustochytriales fatty acid desaturasepromoter. In one aspect, the vector comprises a nucleic acid sequenceselected from the group consisting of SEQ ID NO:18, SEQ ID NO:21, andSEQ ID NO:23.

Yet another embodiment of the present invention relates to a method fortransformation of cells of a microorganism of the OrderThraustochytriales. The method includes a first step of (a) introducinginto cells of a microorganism of the Order Thraustochytriales arecombinant nucleic acid molecule comprising a nucleic acid sequenceencoding an acetolactate synthase that confers onto the cells reducedsensitivity to compounds selected from the group consisting of:sulfonylurea compounds, imidazolinone-class inhibitors, and pyrimidinyloxybenzoates, wherein the acetolactate synthase has an amino acidsequence selected from the group consisting of: (i) an amino acidsequence selected from the group consisting of SEQ ID NO:19, SEQ IDNO:22 and SEQ ID NO:24; and, (ii) an amino acid sequence that is atleast about 75% identical to an amino acid sequence of (i). The methodincludes a second step of (b) selecting cells that have beensuccessfully transformed with the recombinant nucleic acid molecule byculturing the cells of (a) in a medium containing at least one compoundthat is inhibitory to untransformed cells, the compound being selectedfrom the group consisting of: a sulfonylurea compound, animidazolinone-class inhibitor, and pyrimidinyl oxybenzoates. In oneaspect, the nucleic acid sequence encodes an acetolactate synthasehaving an amino acid sequence that is at least about 85% identical, andmore preferably at least about 95% identical, to an amino acid sequenceof (i). In one aspect, the nucleic acid sequence encodes a proteinhaving an amino acid sequence that differs from SEQ ID NO:15 by an aminoacid deletion, insertion, or substitution at an amino acid positionselected from the group consisting of: 116G, 117A, 192P, 200A, 251K,358M, 383D, 592V, 595W, and 599F. In another aspect, the acetolactatesynthase has an amino acid sequence selected from the group consistingof SEQ ID NO:19, SEQ ID NO:22 and SEQ ID NO:24. Preferably, the nucleicacid sequence is selected from the group consisting of: nucleotides1260-3314 of SEQ ID NO:18, nucleotides 1260-3314 of SEQ ID NO:21, andnucleotides 1260-3314 of SEQ ID NO:23. In yet another aspect, thenucleic acid sequence is operatively linked to a transcription controlsequence selected from the group consisting of a Thraustochytrialesα-tubulin promoter, a Thraustochytriales acetolactate synthase promoter,a promoter from a Thraustochytriales polyketide synthase (PKS) system,and a Thraustochytriales fatty acid desaturase promoter.

In one aspect, the recombinant nucleic acid molecule further comprises anucleic acid sequence encoding a protein to be produced by the cell,wherein the nucleic acid sequence encoding the protein is operativelylinked to a transcription control sequence. In one aspect of thisembodiment, the protein is associated with the synthesis of a fatty acidselected from the group consisting of docosahexaenoic acid (DHA),docosapentaenoic acid (DPA), eicosapentaenoic acid (EPA) and arachadonicacid (ARA). In another aspect of this embodiment, the protein isselected from the group consisting of: a fatty acid synthase, a fattyacid desaturase, a fatty acid elongase, a protein associated with apolyketide synthase complex and a protein associated with incorporationof fatty acids into phospholipids or into triacylglycerol molecules. Inone aspect, the protein is an ω-3 fatty acid desaturase is encoded by anucleic acid sequence SEQ ID NO:29. In another aspect, the protein is apolyenoic fatty acid isomerase. In yet another aspect, the protein isselected from the group consisting of HMG-CoA synthase, HMG-CoAreductase, squalene synthase, phytoene synthase, phytoene desaturase, acarotenoid cyclase, a carotenoid hyroxylase, a carotenoid ketolase,vitamin E and lipoic acid.

In another aspect of the present method, the recombinant nucleic acidmolecule in step (a) further comprises a nucleic acid sequence thathybridizes with a target nucleic acid sequence in the microorganism suchthat a gene comprising the target nucleic acid sequence is mutated orinactivated by homologous recombination with the second nucleic acidsequence. In this aspect, the target nucleic acid sequence can encode aprotein selected from the group consisting of lipases, fatty acidoxidation enzymes, proteins involved in carbohydrate synthesis, proteinsinvolved in synthesis of products of isoprenoid pathways, proteinsinvolved in synthesis of cell wall components, proteins involved in thesaturated fatty acid synthesis pathways, proteins involved in thepolyunsaturated fatty acid synthesis pathways, proteins associated witha polyketide synthase complex, and proteins associated withincorporation of fatty acids into phospholipids or triacylglycerolmolecules.

The present method can further include the step of introducing into thecell at least one additional recombinant nucleic acid moleculecomprising a nucleic acid sequence encoding a protein to be expressed,the nucleic acid sequence being operatively linked to a transcriptioncontrol sequence. In another aspect, the method can further include astep of introducing into the cell at least one additional recombinantnucleic acid molecule comprising a second nucleic acid sequence thathybridizes with a target nucleic acid sequence in the microorganism suchthat a gene comprising the target nucleic acid sequence is mutated orinactivated by homologous recombination with the second nucleic acidsequence. In another aspect, the method can further include the step ofintroducing into the cell a recombinant nucleic acid molecule comprisinga nucleic acid sequence encoding a bleomycin-binding protein. In thisaspect, the recombinant nucleic acid molecule comprising a nucleic acidsequence encoding a bleomycin-binding protein further comprises anucleic acid sequence encoding a second protein to be expressed by thecell, wherein the nucleic acid sequence encoding the second protein isoperatively linked to a transcription control sequence. Such atranscription control sequence can include, but is not limited to, aThraustochytriales α-tubulin promoter, a Thraustochytriales acetolactatesynthase promoter, a promoter from a Thraustochytriales polyketidesynthase (PKS) system, and a Thraustochytriales fatty acid desaturasepromoter. In a further aspect of this embodiment, the recombinantnucleic acid molecule comprising a nucleic acid sequence encoding ableomycin-binding protein further comprises a second nucleic acidsequence that hybridizes with a target nucleic acid sequence in themicroorganism such that a gene comprising the target nucleic acidsequence is mutated or inactivated by homologous recombination with thesecond nucleic acid sequence. In one embodiment, the recombinant nucleicacid molecule comprises a nucleic acid sequence SEQ ID NO:9.

In the method of the present invention the microorganism can be from agenus that includes, but is not limited to, Thraustochytrium,Labyrinthuloides, Japonochytrium, and Schizochytrium. In one aspect, themicroorganism is from a species including, but not limited to,Schizochytrium sp., Schizochytrium aggregatum, Schizochytrium limacinum,Thraustochytrium sp., Thraustochytrium striatum, Thraustochytriumaureum, Thraustochytrium roseum, and Japonochytrium sp.

In one embodiment of the present method, the step of introducing isperformed by a method selected from the group consisting of particlebombardment, electroporation, microinjection, lipofection, adsorption,infection and protoplast fusion.

Another embodiment of the present invention relates to a recombinantmicroorganism of the order Thraustochytriales, transformed with arecombinant nucleic acid molecule comprising a nucleic acid sequenceencoding an acetolactate synthase that confers onto the microorganismreduced sensitivity to compounds selected from the group consisting of:sulfonylurea compounds, imidazolinone-class inhibitors, and pyrimidinyloxybenzoates. The acetolactate synthase has an amino acid sequenceselected from the group consisting of: (a) an amino acid sequenceselected from the group consisting of SEQ ID NO:19, SEQ ID NO:22 and SEQID NO:24; and, (b) an amino acid sequence that is at least about 75%identical to an amino acid sequence of (a). In one aspect, the nucleicacid sequence encodes an acetolactate synthase having an amino acidsequence that is at least about 85% identical to an amino acid sequenceof (a). In another aspect, the nucleic acid sequence encodes anacetolactate synthase having an amino acid sequence that is at leastabout 95% identical to an amino acid sequence of (a). In another aspect,the acetolactate synthase has an amino acid sequence selected from thegroup consisting of SEQ ID NO:19, SEQ ID NO:22 and SEQ ID NO:24. In yetanother aspect, the nucleic acid sequence is selected from the groupconsisting of: nucleotides 1260-3314 of SEQ ID NO:18, nucleotides1260-3314 of SEQ ID NO:21, and nucleotides 1260-3314 of SEQ ID NO:23. Inyet another aspect, the recombinant nucleic acid molecule comprises anucleic acid sequence selected from the group consisting of SEQ IDNO:18, SEQ ID NO:21 and SEQ ID NO:23. Preferably, the nucleic acidsequence encoding an acetolactate synthase is operatively linked to apromoter that functions in a Thraustochytriales microorganism. In oneaspect, the nucleic acid sequence encoding an acetolactate synthase isoperatively linked to a transcription control sequence selected from thegroup consisting of a Thraustochytriales α-tubulin promoter, aThraustochytriales acetolactate synthase promoter, a promoter from aThraustochytriales polyketide synthase (PKS) system, and aThraustochytriales fatty acid desaturase promoter. In one embodiment,the recombinant nucleic acid molecule further comprises a nucleic acidsequence encoding a first protein for production by the microorganism,wherein the nucleic acid sequence encoding the first protein isoperatively linked to a transcription control sequence. In anotherembodiment, the recombinant cell is further transformed with arecombinant nucleic acid molecule comprising a nucleic acid sequenceencoding a bleomycin-binding protein. Preferably, the recombinantnucleic acid molecule comprises a nucleic acid sequence SEQ ID NO:9. Inone embodiment, the recombinant nucleic acid molecule comprising anucleic acid sequence encoding a bleomycin-binding protein furthercomprises a nucleic acid sequence encoding a second protein forproduction by the cell, wherein the nucleic acid sequence encoding thesecond protein is operatively linked to a transcription controlsequence. In one embodiment, the microorganism also includes at leastone additional recombinant nucleic acid molecule comprising a nucleicacid sequence encoding a protein for production by the cell.

Yet another embodiment of the present invention relates to an isolatednucleic acid molecule comprising a nucleic acid sequence selected fromthe group consisting of: (a) nucleotides 441-894 of SEQ ID NO:9; (b) anucleic acid sequence that is at least about 95% identical tonucleotides 441-894 of SEQ ID NO:9 over the full length of nucleotides441-894 of SEQ ID NO:9, wherein the nucleic acid sequence has at leastbasal α-tubulin promoter transcriptional activity; and (c) an isolatedpolynucleotide comprising a nucleic acid sequence that is fullycomplementary to the polynucleotide of (a) or (b). Preferably, theisolated nucleic acid molecule comprises a nucleic acid sequenceselected from the group consisting of SEQ ID NO:4 and nucleotides441-894 of SEQ ID NO:9.

Yet another embodiment of the present invention relates to a recombinantvector for transformation of microorganisms of the OrderThraustochytriales, comprising a nucleic acid sequence encoding ableomycin binding protein operatively linked to a promoter selected fromthe group consisting of a Thraustochytriales α-tubulin promoter, aThraustochytriales acetolactate synthase promoter, a promoter from aThraustochytriales polyketide synthase (PKS) system, and aThraustochytriales fatty acid desaturase promoter. In one aspect, theThraustochytriales acetolactate synthase promoter comprises nucleotides1-1259 of SEQ ID NO:14. In one aspect, the α-tubulin promoter comprisesa nucleic acid sequence selected from the group consisting ofnucleotides 441-894 of SEQ ID NO:9, and a nucleic acid sequence that isat least about 95% identical to nucleotides 441-894 of SEQ ID NO:9 overthe full length of nucleotides 441-894 of SEQ ID NO:9, wherein thenucleic acid sequence has at least basal α-tubulin promotertranscriptional activity. In another aspect, a promoter from aThraustochytriales PKS system comprises SEQ ID NO:34 or a nucleic acidsequence contained within SEQ ID NO:34, wherein said promoter has atleast basal PKS promoter transcriptional activity. In another aspect,the recombinant vector comprises a nucleic acid sequence selected fromthe group consisting of SEQ ID NO:8 and SEQ ID NO:9.

BRIEF DESCRIPTION OF THE DRAWINGS

FIG. 1 illustrates the construction of recombinant plasmid pTUBZEO-11.

FIG. 2 illustrates the construction of recombinant plasmid pTUBZEO11-2.

FIG. 3A illustrates recombinant plasmid pMON50200.

FIG. 3B illustrates recombinant plasmid pMON50201.

FIG. 3C illustrates recombinant plasmid pMON50202.

FIG. 3D illustrates recombinant plasmid pMON50203.

DETAILED DESCRIPTION OF THE INVENTION

This invention comprises methods and related materials to geneticallytransform microorganisms of the order Thraustochytriales. All of thestrains of unicellular microorganisms disclosed herein for use as atransformant of the recombinant constructs of the present invention,which can generally also be referred to as Thraustochytrids, are membersof the order Thraustochytriales. According to the present invention, thephrases “Thraustochytrid”, “Thraustochytriales microorganism” and“microorganism of the order Thraustochytriales” can be usedinterchangeably. The present inventors are not aware of any priorreports that describe a transformation system for Schizochytrium or anyother Thraustochytriales microorganism. The transformation systemsdescribed herein can be used to introduce foreign genes intomicroorganisms of the order Thraustochytriales, thereby providing ameans to create strains with enhanced commercial value. In addition,this invention enables the mutation or inactivation of specific genes byhomologous or nonhomologous recombination, providing a new way to altercellular metabolism and to identify the functions of specific genes inThraustochytriales microorganisms.

More specifically, the present inventors have demonstrated genetictransfoiination of a Thraustochytriales microorganism of the genus,Schizochytrium (Order: Thraustochytriales; Family: Thraustochytriaceae;Genus: Schizochytrium), by the use of two types of transformationvectors. These vectors can be introduced into cells by standard methods,followed by identification and isolation of recombinant cells based ontheir ability to grow in the presence of selective compounds. Thepresent inventors have demonstrated the effectiveness of these vectorsby introducing them via particle bombardment, but other means tointroduce the vectors can also be used (e.g., electroporation) and areknown in the art and are intended to be encompassed by the presentinvention.

For one transformation vector, exemplified herein by the recombinantvector denoted pTUBZEO11-2, a chimeric gene was created in which the blegene (which encodes a “bleomycin-binding protein”) fromStreptoalloteichus hindustanus was placed downstream from aSchizochytrium tubulin gene promoter. An SV40 terminator was placeddownstream from the ble gene in this construct. This vector enablesexpression of the ble gene in Schizochytrium, thereby conferringresistance to Zeocin™ and related compounds, which are toxic towild-type cells when included in the growth medium at appropriatelevels. The source of the ble gene and SV40 terminator in this constructwas a commercially available vector, named pSV40/Zeo, which was acquiredfrom Invitrogen Corporation (Carlsbad, Calif.) (Technical Manual 180202,Version B, “ZeoCassette Vectors”; Invitrogen Corporation, 1600 FaradayAve., Carlsbad, Calif. 92008). The tubulin gene promoter was isolatedvia the polymerase chain reaction; one of the primers used for thereaction was based on sequence data obtained through a randomSchizochytrium cDNA sequencing project. The map of pTUBZEO11-2 is shownin FIG. 2, and the nucleotide sequence of pTUBZEO11-2 is represented bySEQ ID NO:9. Transformation of Schizochytrium with this vector wasconfirmed by the use of the polymerase chain reaction and Southern blotanalysis to detect the presence of vector sequences integrated into theSchizochytrium genome.

The ble gene has been used by prior investigators as a selectable markerfor genetic transformation of a variety of organisms, includingbacteria, non-Thraustochytrid microalgae, fungi, protozoa, plants, andanimal cells (See, for example, U.S. Pat. No. 6,027,900; Lumbreras etal., 1998, Plant J. 14:441-447; Rohe et al., 1996, Curr. Genet.29:587-590; Messina et al., 1995, Gene 165:213-217; Guerrero et al.,1992, Appl. Microbiol. Biotechnol. 36:759-762; Perez et al., 1989, PlantMol. Biol. 13:365-373; Gatigno et al., 1990 Gene 91:35-41). The ble geneencodes a “bleomycin-binding protein” that confers resistance to severalantibiotics, including bleomycin, phleomycin, and Zeocin™ (Drocourt etal., 1990, Nucleic Acids Res. 18:4009). This gene is availablecommercially from Invitrogen Corporation, which was the source of thegene that the present inventors used for creating the Schizochytriumtransformation vector pTUBZEO11-2. However, the present inventors arebelieved to be the first to produce a transformation vector in which theble gene is attached to a Thraustochytrid promoter in a manner thatallows expression of the gene in Thraustochytrids.

A second set of transformation vectors was created by in vitrosite-directed mutagenesis of an acetolactate synthase gene (als) thatthe present inventors isolated from a Schizochytrium genomic library.These mutations change the amino acid sequence of the encoded enzyme(ALS) in such a way that it is much less sensitive to sulfometuronmethyl and other sulfonylurea compounds, as well as imidazolinone-classinhibitors and pyrimidinyl oxybenzoates, to which microorganisms of theorder Thraustochytriales are sensitive. Sulfonylurea compounds such assulfometuron methyl (SMM) are often toxic to cells because they are ableto bind to and inactivate the enzyme acetolactate synthase (ALS) from avariety of organisms. ALS catalyzes the first step in the biosynthesisof the amino acids valine, leucine, and isoleucine. Imidazolinones,triazolopyrimidines, and other compounds have also been shown to bind toand inactivate ALS from certain organisms. Mutant forms of genes thatencode acetolactate synthase (also known as acetohydroxy acid synthase)from other organisms have been used previously as selectable markers fortransformation of yeast and plants (Annu. Rev. Plant Physiol. Plant Mol.Biol. 40:441-470, 1989). However, there are no reports prior to thepresent invention that describe the sequence or properties of the alsgene from Schizochytrium or any other Thraustochytriales member, or theuse of mutant Thraustochytriales als genes to confer resistance tosulfonylurea, imidazolinone or pyrimidinyl oxybenzoate compounds. Infact, to the present inventors' knowledge, there have not even been anypublished reports regarding the sensitivity of Thraustochytrialesmicroorganisms to these selective agents, including sulfometuron methyl,and therefore, it was not known prior to the present invention whethersuch a selectable marker would even be feasible for use in aThraustochytrid transformation system. It is noteworthy that genes withsubstantial homology to known als genes occur in various organisms, butdo not encode enzymes that are able to catalyze the synthesis ofacetolactate (Biochimica et Biophysica Acta 1385:401-419, 1998).Therefore, it would not have been obvious that a cloned als homologue infact encodes ALS. In order to definitively determine whether the clonedSchizochytrium gene was a true als gene, the present inventorsdemonstrated, through transformation experiments, a positive correlationof sulfometuron methyl-resistance with expression of the mutatedputative Schizochytrium als gene.

The present inventors have produced three different transformationvectors containing mutant als genes: one mutant als gene encodes anenzyme with a valine at position 595 instead of a tryptophan (plasmidpMON50201, or ALSmut1-7), another encodes a glutamine at position 192instead of a proline (plasmid pMON50202, or ALSmut2-2), and a third formcontains both of these mutations (plasmid pMON50203, or ALSmut3-5). Inthese vectors, the expression of the recombinant mutant als genes isunder the control of the native als gene promoter and terminator. Themaps of these vectors, along with a vector containing the wild-typeSchizochytrium als gene (plasmid pMON50200, or AE-5), are shown in FIGS.3A-3D. Transformation of Schizochytrium with these mutant ALS-encodingvectors was confirmed by the use of the polymerase chain reaction andSouthern blot analysis to detect the presence of vector sequencesintegrated into the Schizochytrium genome. As described in detail below,now that the present inventors have identified the complete sequence forthe als gene, other mutations, specified below, can also be made.Therefore, the described mutant als genes are intended to be exemplary,and not inclusive of all possible mutations.

The transformation systems of the present invention have been used tointroduce foreign genes into Thraustochytriales cells viacotransformation. In these cases, the foreign genes were placed betweenvarious Schizochytrium promoters and an appropriate terminator (e.g.,SV40 or a Schizochytrium gene terminator region). For example, thepresent inventors have produced and introduced a synthetic gene thatencodes an ω-3 fatty acid desaturase from the nematode Caenorhabditiselegans, represented herein by SEQ ID NO:29, to increase the levels ofdocosahexaenoic acid in Schizochytrium. SEQ ID NO:30 represents theamino acid sequence of the desaturase encoded by SEQ ID NO:29.Expression cassettes containing foreign genes can also be introducedinto Thraustochytriales cells by direct inclusion within the selectablemarker-containing transformation vector.

Moreover, the present inventors have also demonstrated with the mutantALS-encoding vectors that homologous recombination occurs inSchizochytrium, indicating the feasibility of using recombinant means toinactivate or mutate specific native Schizochytrium genes.

With regard to the Thraustochytriales promoter sequences describedherein, a sequence database search (GenBank) for all nucleotide andprotein sequences reported for members of the order Thraustochytriales,indicates that as of the time of the present invention, no promotersequences from Schizochytrium or any other member of Thraustochytrialeshave been reported. The only gene that has been reported from anySchizochytrium species is for the 5S ribosomal RNA of S. aggregatum(GenBank accession numbers X06104 and M13616). 5S and 18S ribosomal RNAsequences have been reported for the Thraustochytriales members, speciesUlkenia, and genera Labyrinthuloides and Thraustochytrium, but this hasno bearing on the present invention. A partial coding region of a“putative T3/T7-like RNA polymerase” gene from Thraustochytrium aureumhas been described (Nucleic Acids Research 15:648-654, 1996), but apromoter sequence for this gene was not reported.

This invention can be used to introduce any genes or other nucleotidesequences that are of interest into a microorganism of the orderThraustochytriales. Such nucleotide sequences include, but are notlimited to, nucleic acids encoding proteins (e.g., enzymes) associatedwith the synthesis of fatty acids (e.g., the fatty acids:docosahexaenoic acid (DHA), docosapentaenoic acid (DPA),eicosapentaenoic acid (EPA) and arachadonic acid (ARA). Such proteinsinclude, but are not limited to: fatty acid synthases, fatty aciddesaturases, and fatty acid elongases, as well as proteins associatedwith a polyketide synthase complex and/or proteins associated withincorporation of such fatty acids into phospholipids or intotriacylglycerol molecules. For example, the invention has been used tointroduce genes encoding various ω-3 fatty acid desaturases intoSchizochytrium in an attempt to increase the level of docosahexaenoicacid (DHA) in the cells via ω-3 desaturation of docosapentaenoic acid(DPA). As another example, expression of a putative polyenoic fatty acidisomerase from the red alga, Ptilota, in Schizochytrium has also beendemonstrated. The genes encoding a Schizochytrium polyketide synthasecomplex (i.e., a polyketide synthase system) have been deposited asGenBank Accession Nos. AF378329 (ORFA), AF378328 (ORFB) and AF378329(ORFC).

The present invention is also useful for introducing intoThraustochytriales microorganisms genes and other nucleotide sequencesencoding proteins associated with the isoprenoid biosynthetic pathway.Such proteins include, but are not limited to, HMG-CoA synthase andHMG-CoA reductase. Other suitable proteins include proteins associatedwith the synthesis of molecules derived from isoprenoid subunitsincluding, but not limited to, various steroid compounds and variouscarotenoid compounds. Proteins associated with the synthesis of variouscarotenoid compounds include, but are not limited to, squalene synthase,phytoene synthase, phytoene desaturase, and various carotenoid cyclases,hydroxylases and ketolases.

The present invention is also useful for introducing intoThraustochytriales one or more nucleic acid sequences encoding proteinsassociated with the synthesis of anti-oxidant compounds including, butnot limited to, vitamin E and lipoic acid.

In addition, the present invention can be used to introduce any genes orother nucleotide sequences vectors into Thraustochytrialesmicroorganisms in order to inactivate or delete genes (i.e., “knock-out”or “targeted gene disruption”). The inactivation or deletion of genes istypically used for the purpose of enhancing the commercial value of themicroorganism. For example, it may be desirable to remove genes thatencode enzymes (or nucleic acids which regulate the expression of suchgenes) of the saturated and polyunsaturated fatty acid synthesispathways. In another aspect, it may be desirable to inhibit or knock-outgenes encoding proteins that are involved in the degradation of othervaluable compounds produced by the Thraustochytriales microorganism orwhich otherwise lessen the value of the desired compound. For example,genes encoding lipases, fatty acid oxidation enzymes, and proteins thathave objectionable flavors or odors may be desirable knock-out targetsby the present invention. In yet another aspect, it may be desirable toknock-out genes encoding proteins that are associated with the synthesisof compounds whose synthesis is in competition with other molecules ofinterest. For example, such genes include, but are not limited to, genesencoding proteins involved in carbohydrate biosynthesis, genes encodingproteins involved in the synthesis of various products of isoprenoidpathways (e.g., sterols or specific carotenoid compounds), and genesencoding proteins involved in the synthesis of cell wall components. Byway of example, genes have been introduced into Schizochytrium cells bythe use of this invention in an attempt to inactivate genes that arehomologous to the polyketide synthase genes from Shewanella in order toassess their role in the production of highly unsaturated fatty acids(HUFA). As exemplified by Example 6, the present invention can also beused to inactivate, delete, or mutate native genes that are involved inthe production of fatty acids, carotenoids, sterols, vitamins, or othercompounds in order to improve the economics or acceptability of productsthat are related to these compounds. It is noted that in someembodiments, as discussed above, it may be desirable to enhanceproduction of a given protein, whereas in other embodiments, it may bedesirable to inhibit production of the same protein. Such determinationsare based on the given use and production goals for a specificmicroorganism. The present invention is also useful for determining theprocess of genetic recombination in Schizochytrium.

Other genes and nucleic acid molecules useful for introduction intoThraustochytriales will be apparent to those of skill in the art, andall such genes and molecules are intended to be encompassed by thepresent invention.

Various embodiments of the present invention are described belowinitially with regard to a Thraustochytriales als gene and/or ALSprotein of the present invention. It is to be understood, however, thatthe general definitions of terms and methods are intended to apply tothe discussion of other genes, nucleic acids and proteins describedbelow.

The present invention is based in part on the identification, isolationand production of nucleic acid sequences encoding selectable markersthat are suitable for use in recombinant constructs for thetransformation of Thraustochytrid microorganisms. Such selectablemarkers allow the selection of microorganisms that have beensuccessfully transformed with the recombinant constructs of the presentinvention. One selectable marker useful for the transformation ofThraustochytriales according to the present invention is aThraustochytriales acetolactate synthase (i.e., ALS). Preferably, theacetolactate synthase has been modified, mutated, or otherwise selected,to be resistant to inhibition by sulfonylurea compounds,imidazolinone-class inhibitors and/or pyrimidinyl oxybenzoates (i.e.,such an ALS is a homologue of a naturally occurring acetolactatesynthase).

According to the present invention, an acetolactate synthase is aprotein that has acetolactate synthase biological activity, includingfull-length proteins, fusion proteins, or any homologue of a naturallyoccurring acetolactate synthase. A homologue of an acetolactate synthaseincludes proteins which differ from a naturally occurring acetolactatesynthase in that at least one or a few, but not limited to one or a few,amino acids have been deleted (e.g., a truncated version of the protein,such as a peptide or fragment), inserted, inverted, substituted and/orderivatized (e.g., by glycosylation, phosphorylation, acetylation,myristoylation, prenylation, palmitation, amidation and/or addition ofglycosylphosphatidyl inositol). Preferred homologues of a naturallyoccurring acetolactate synthase are described in detail below.

An isolated protein, such as an isolated acetolactate synthase,according to the present invention, is a protein that has been removedfrom its natural milieu (i.e., that has been subject to humanmanipulation) and can include purified proteins, partially purifiedproteins, recombinantly produced proteins, and synthetically producedproteins, for example. As such, “isolated” does not reflect the extentto which the protein has been purified. Preferably, an isolatedacetolactate synthase of the present invention is producedrecombinantly. A “Thraustochytriales acetolactate synthase” refers to anacetolactate synthase (including a homologue of a naturally occurringacetolactate synthase) from a Thraustochytriales microorganism or thathas been otherwise produced from the knowledge of the structure (e.g.,sequence) of a naturally occurring acetolactate synthase from aThraustochytriales microorganism. In other words, a Thraustochytrialesacetolactate synthase includes any acetolactate synthase that has thestructure and function of a naturally occurring acetolactate synthasefrom a Thraustochytriales microorganism or that is a biologically active(i.e., has biological activity) homologue of a naturally occurringacetolactate synthase from a Thraustochytriales microorganism asdescribed in detail herein. As such, a Thraustochytriales acetolactatesynthase can include purified, partially purified, recombinant,mutated/modified and synthetic proteins.

In general, the biological activity or biological action of a proteinrefers to any function(s) exhibited or performed by the protein that isascribed to the naturally occurring form of the protein as measured orobserved in vivo (i.e., in the natural physiological environment of theprotein) or in vitro (i.e., under laboratory conditions). For example, abiological activity of an acetolactate synthase includes acetolactatesynthase enzymatic activity. Modifications of a protein, such as in ahomologue or mimetic (discussed below), may result in proteins havingthe same biological activity as the naturally occurring protein, or inproteins having decreased or increased biological activity as comparedto the naturally occurring protein. Modifications which result in adecrease in protein expression or a decrease in the activity of theprotein, can be referred to as inactivation (complete or partial),down-regulation, or decreased action of a protein. Similarly,modifications which result in an increase in protein expression or anincrease in the activity of the protein, can be referred to asamplification, overproduction, activation, enhancement, up-regulation orincreased action of a protein.

With regard to the acetolactate synthase of the present invention, it ispreferred that modifications present in acetolactate synthasehomologues, as compared to a naturally occurring acetolactate synthase,do not substantially change, or at least do not substantially decrease,the basic biological activity of the synthase as compared to thenaturally occurring protein. However, such homologues may havedifferences in characteristics other than the functional, or enzymatic,activity of the protein as compared to the naturally occurring form,such as a decreased sensitivity to inhibition by certain compounds ascompared to the naturally occurring protein. Preferably, a homologue ofa naturally occurring acetolactate synthase has reduced (i.e.,decreased, lessened) sensitivity to compounds that bind to andinactivate naturally occurring acetolactate synthases as compared to thenaturally occurring acetolactate synthase from which the homologue wasderived. For example, sulfonylurea compounds, such as sulfometuronmethyl (SMM), are often toxic to cells because they are able to bind toand inactivate acetolactate synthase (ALS). Imidazolinones,triazolopyrimidines, and other similar compounds (referred to generallyherein as imidazolinone-class inhibitors) have also been shown to bindto and inactivate ALS. Therefore, a homologue of a naturally occurringacetolactate synthase preferably has reduced sensitivity to sulfonylureacompounds, as well as to imidazolinone-class inhibitors (e.g., by havingdisrupted binding sites for such inhibitors or binding sites withreduced affinity for the inhibitor) and to pyrimidinyl oxybenzoates,while maintaining acetolactate synthase enzymatic activity.

As used herein, a protein that has “acetolactate synthase biologicalactivity” or that is referred to as an “acetolactate synthase” refers toa protein that catalyzes the first step in the biosynthesis of the aminoacids valine, leucine, and isoleucine. More specifically, an isolatedacetolactate synthase of the present invention, including full-lengthproteins, truncated proteins, fusion proteins and homologues, can beidentified in a straight-forward manner by the proteins' ability tocatalyze the synthesis of acetolactate from pyruvate. Acetolactatesynthase biological activity can be evaluated by one of skill in the artby any suitable in vitro or in vivo assay for enzyme activity.

In one embodiment, an acetolactate synthase of the present invention hasan amino acid sequence that is at least about 65% identical to an aminoacid sequence of selected from the group of SEQ ID NO:15, SEQ ID NO:19,SEQ ID NO:22, SEQ ID NO:24, over at least about 600 amino acids of anyof such sequences, wherein the protein is an acetolactate synthase(i.e., has acetolactate synthase biological activity). More preferably,an acetolactate synthase of the present invention has an amino acidsequence that is at least about 70% identical, and more preferably, atleast about 75% identical, and even more preferably at least about 80%identical, and even more preferably at least about 85% identical, andeven more preferably at least about 90% identical and even morepreferably at least about 95% identical, and even more preferably atleast about 96% identical, and even more preferably at least about 97%identical, and even more preferably at least about 98% identical, andeven more preferably at least about 99% identical to any of SEQ IDNO:15, SEQ ID NO:19, SEQ ID NO:22 or SEQ ID NO:24, over at least about600 amino acids of any of SEQ ID NO:15, SEQ ID NO:19, SEQ ID NO:22 orSEQ ID NO:24, wherein the protein has acetolactate synthase biologicalactivity.

In another embodiment, an acetolactate synthase of the present inventionhas an amino acid sequence that is at least about 75% identical to anamino acid sequence of selected from the group of SEQ ID NO:15, SEQ IDNO:19, SEQ ID NO:22, SEQ ID NO:24, over at least 95 amino acids of anyof such sequences, wherein the protein is an acetolactate synthase(i.e., has acetolactate synthase biological activity). More preferably,an acetolactate synthase of the present invention has an amino acidsequence that is at least about 80% identical, and even more preferablyat least about 85% identical, and even more preferably at least about90% identical and even more preferably at least about 95% identical, andeven more preferably at least about 96% identical, and even morepreferably at least about 97% identical, and even more preferably atleast about 98% identical, and even more preferably at least about 99%identical to any of SEQ ID NO:15, SEQ ID NO:19, SEQ ID NO:22 or SEQ IDNO:24, over at least 95 amino acids of any of SEQ ID NO:15, SEQ IDNO:19, SEQ ID NO:22 or SEQ ID NO:24, wherein the protein hasacetolactate synthase biological activity. Even more preferably, anacetolactate synthase of the present invention has an amino acidsequence that has any of the above-referenced percent identities to anyof SEQ ID NO:15, SEQ ID NO:19, SEQ ID NO:22 or SEQ ID NO:24 over atleast 100 amino acids, and more preferably 125, and more preferably 150,and more preferably 175, and more preferably 200, and more preferably225, and more preferably 250, and more preferably 275, and morepreferably 300, and more preferably 325, and more preferably 350, andmore preferably 375, and more preferably 400, and more preferably 425,and more preferably 450, and more preferably 475, and more preferably500, and more preferably 525, and more preferably 550, and morepreferably 575 amino acids of any of SEQ ID NO:15, SEQ ID NO:19, SEQ IDNO:22 or SEQ ID NO:24, wherein the protein has acetolactate synthasebiological activity.

As used herein, unless otherwise specified, reference to a percent (%)identity refers to an evaluation of homology which is performed using:(1) a BLAST 2.0 Basic BLAST homology search using blastp for amino acidsearches and blastn for nucleic acid searches with standard defaultparameters, wherein the query sequence is filtered for low complexityregions by default (described in Altschul, S. F., Madden, T. L.,Schääffer, A. A., Zhang, J., Zhang, Z., Miller, W. & Lipman, D. J.(1997) “Gapped BLAST and PSI-BLAST: a new generation of protein databasesearch programs.” Nucleic Acids Res. 25:3389-3402, incorporated hereinby reference in its entirety); (2) a BLAST 2 alignment (using theparameters described below); (3) and/or PSI-BLAST with the standarddefault parameters (Position-Specific Iterated BLAST. It is noted thatdue to some differences in the standard parameters between BLAST 2.0Basic BLAST and BLAST 2, two specific sequences might be recognized ashaving significant homology using the BLAST 2 program, whereas a searchperformed in BLAST 2.0 Basic BLAST using one of the sequences as thequery sequence may not identify the second sequence in the top matches.In addition, PSI-BLAST provides an automated, easy-to-use version of a“profile” search, which is a sensitive way to look for sequencehomologues. The program first performs a gapped BLAST database search.The PSI-BLAST program uses the information from any significantalignments returned to construct a position-specific score matrix, whichreplaces the query sequence for the next round of database searching.Therefore, it is to be understood that percent identity can bedetermined by using any one of these programs.

Two specific sequences can be aligned to one another using BLAST 2sequence as described in Tatusova and Madden, (1999), “Blast 2sequences—a new tool for comparing protein and nucleotide sequences”,FEMS Microbiol Lett. 174:247-250, incorporated herein by reference inits entirety. BLAST 2 sequence alignment is performed in blastp orblastn using the BLAST 2.0 algorithm to perform a Gapped BLAST search(BLAST 2.0) between the two sequences allowing for the introduction ofgaps (deletions and insertions) in the resulting alignment. For purposesof clarity herein, a BLAST 2 sequence alignment is performed using thestandard default parameters as follows.

For blastn, using 0 BLOSUM62 matrix:

Reward for match=1

Penalty for mismatch=−2

Open gap (5) and extension gap (2) penalties

gap x_dropoff (50) expect (10) word size (11) filter (on)

For blastp, using 0 BLOSUM62 matrix:

Open gap (11) and extension gap (1) penalties

gap x_dropoff (50) expect (10) word size (3) filter (on).

An acetolactate synthase of the present invention can also includeproteins having an amino acid sequence comprising at least 30 contiguousamino acid residues of any of SEQ ID NO:15, SEQ ID NO:19, SEQ ID NO:22or SEQ ID NO:24, (i.e., 30 contiguous amino acid residues having 100%identity with 30 contiguous amino acids of any of SEQ ID NO:15, SEQ IDNO:19, SEQ ID NO:22 or SEQ ID NO:24). In a preferred embodiment, anacetolactate synthase of the present invention includes proteins havingamino acid sequences comprising at least 50, and more preferably atleast 75, and more preferably at least 100, and more preferably at least115, and more preferably at least 130, and more preferably at least 150,and more preferably at least 200, and more preferably, at least 250, andmore preferably, at least 300, and more preferably, at least 350, andmore preferably, at least 400, and more preferably, at least 450, andmore preferably, at least 500, and more preferably, at least 550, andmore preferably, at least 600, and more preferably, at least 650,contiguous amino acid residues of any of SEQ ID NO:15, SEQ ID NO:19, SEQID NO:22 or SEQ ID NO:24. Such a protein has acetolactate synthasebiological activity.

According to the present invention, the term “contiguous” or“consecutive”, with regard to nucleic acid or amino acid sequencesdescribed herein, means to be connected in an unbroken sequence. Forexample, for a first sequence to comprise 30 contiguous (or consecutive)amino acids of a second sequence, means that the first sequence includesan unbroken sequence of 30 amino acid residues that is 100% identical toan unbroken sequence of 30 amino acid residues in the second sequence.Similarly, for a first sequence to have “100% identity” with a secondsequence means that the first sequence exactly matches the secondsequence with no gaps between nucleotides or amino acids.

In another embodiment, an acetolactate synthase of the presentinvention, including an acetolactate synthase homologue, includes aprotein having an amino acid sequence that is sufficiently similar to anaturally occurring acetolactate synthase amino acid sequence that anucleic acid sequence encoding the homologue is capable of hybridizingunder moderate, high, or very high stringency conditions (describedbelow) to (i.e., with) a nucleic acid molecule encoding the naturallyoccurring acetolactate synthase (i.e., to the complement of the nucleicacid strand encoding the naturally occurring acetolactate synthase aminoacid sequence). Preferably, an acetolactate synthase is encoded by anucleic acid sequence that hybridizes under moderate, high or very highstringency conditions to the complement of a nucleic acid sequence thatencodes a protein comprising an amino acid sequence represented by SEQID NO:15, SEQ ID NO:19, SEQ ID NO:22 or SEQ ID NO:24. Even morepreferably, an acetolactate synthase of the present invention is encodedby a nucleic acid sequence that hybridizes under moderate, high or veryhigh stringency conditions to the complement of nucleotides 1260-3314 ofSEQ ID NO:15, nucleotides 1260-3314 of SEQ ID NO:18, nucleotides1260-3314 of SEQ ID NO:21, or nucleotides 1260-3314 of SEQ ID NO:23.Such hybridization conditions are described in detail below. A nucleicacid sequence complement of nucleic acid sequence encoding anacetolactate synthase of the present invention refers to the nucleicacid sequence of the nucleic acid strand that is complementary to thestrand which encodes the acetolactate synthase. It will be appreciatedthat a double stranded DNA which encodes a given amino acid sequencecomprises a single strand DNA and its complementary strand having asequence that is a complement to the single strand DNA. As such, nucleicacid molecules of the present invention can be either double-stranded orsingle-stranded, and include those nucleic acid molecules that formstable hybrids under stringent' hybridization conditions with a nucleicacid sequence that encodes the amino acid sequence SEQ ID NO:15, SEQ IDNO:19, SEQ ID NO:22 or SEQ ID NO:24, and/or with the complement of thenucleic acid sequence that encodes any of such amino acid sequences.Methods to deduce a complementary sequence are known to those skilled inthe art. It should be noted that since amino acid sequencing and nucleicacid sequencing technologies are not entirely error-free, the sequencespresented herein, at best, represent apparent sequences of anacetolactate synthase of the present invention.

Acetolactate synthase homologues can be the result of natural allelicvariation or natural mutation. Acetolactate synthase homologues of thepresent invention can also be produced using techniques known in the artincluding, but not limited to, direct modifications to the protein ormodifications to the gene encoding the protein using, for example,classic or recombinant DNA techniques to effect random or targetedmutagenesis. A naturally occurring allelic variant of a nucleic acidencoding an acetolactate synthase is a gene that occurs at essentiallythe same locus (or loci) in the genome as the gene which encodes anamino acid sequence SEQ ID NO:15, but which, due to natural variationscaused by, for example, mutation or recombination, has a similar but notidentical sequence. Natural allelic variants typically encode proteinshaving similar activity to that of the protein encoded by the gene towhich they are being compared. One class of allelic variants can encodethe same protein but have different nucleic acid sequences due to thedegeneracy of the genetic code. Allelic variants can also comprisealterations in the 5′ or 3′ untranslated regions of the gene (e.g., inregulatory control regions). Allelic variants are well known to thoseskilled in the art.

Acetolactate synthase proteins of the present invention also includeexpression products of gene fusions (for example, used to overexpresssoluble, active forms of the recombinant protein), of mutagenized genes(such as genes having codon modifications to enhance gene transcriptionand translation), and of truncated genes (such as genes having membranebinding domains removed to generate soluble forms of a membrane protein,or genes having signal sequences removed which are poorly tolerated in aparticular recombinant host).

The minimum size of a protein and/or homologue of the present inventionis a size sufficient to have acetolactate synthase biological activity.Preferably, a protein of the present invention is at least 30 aminoacids long, and more preferably, at least about 50, and more preferablyat least 75, and more preferably at least 100, and more preferably atleast 115, and more preferably at least 130, and more preferably atleast 150, and more preferably at least 200, and more preferably, atleast 250, and more preferably, at least 300, and more preferably, atleast 350, and more preferably, at least 400, and more preferably, atleast 450, and more preferably, at least 500, and more preferably, atleast 550, and more preferably, at least 600, and more preferably, atleast 650, and more preferably, at least 684 amino acids long. There isno limit, other than a practical limit, on the maximum size of such aprotein in that the protein can include a portion of an acetolactatesynthase protein or a full-length acetolactate synthase, plus additionalsequence (e.g., a fusion protein sequence), if desired.

The present invention also includes a fusion protein that includes anacetolactate synthase-containing domain (i.e., an amino acid sequencefor an acetolactate synthase according to the present invention)attached to one or more fusion segments. Suitable fusion segments foruse with the present invention include, but are not limited to, segmentsthat can: enhance a protein's stability; provide other desirablebiological activity; and/or assist with the purification of anacetolactate synthase (e.g., by affinity chromatography). A suitablefusion segment can be a domain of any size that has the desired function(e.g., imparts increased stability, solubility, action or biologicalactivity; and/or simplifies purification of a protein). Fusion segmentscan be joined to amino and/or carboxyl termini of the acetolactatesynthase-containing domain of the protein and can be susceptible tocleavage in order to enable straight-forward recovery of an acetolactatesynthase. Fusion proteins are preferably produced by culturing arecombinant cell transfected with a fusion nucleic acid molecule thatencodes a protein including the fusion segment attached to either thecarboxyl and/or amino terminal end of an acetolactatesynthase-containing domain.

The present invention also includes a mimetic of an acetolactatesynthase. As used herein, the term “mimetic” is used to refer to anypeptide or non-peptide compound that is able to mimic the biologicalaction of a naturally occurring peptide, often because the mimetic has abasic structure that mimics the basic structure of the naturallyoccurring peptide and/or has the salient biological properties of thenaturally occurring peptide. Mimetics can include, but are not limitedto: peptides that have substantial modifications from the prototype suchas no side chain similarity with the naturally occurring peptide (suchmodifications, for example, may decrease its susceptibility todegradation); anti-idiotypic and/or catalytic antibodies, or fragmentsthereof; non-proteinaceous portions of an isolated protein (e.g.,carbohydrate structures); or synthetic or natural organic molecules,including nucleic acids and drugs identified through combinatorialchemistry, for example.

Such mimetics can be designed, selected and/or otherwise identifiedusing a variety of methods known in the art. Various methods of drugdesign, useful to design mimetics or other therapeutic compounds usefulin the present invention are disclosed in Maulik et al., 1997, MolecularBiotechnology: Therapeutic Applications and Strategies, Wiley-Liss,Inc., which is incorporated herein by reference in its entirety. Anacetolactate synthase mimetic can be obtained, for example, frommolecular diversity strategies (a combination of related strategiesallowing the rapid construction of large, chemically diverse moleculelibraries), libraries of natural or synthetic compounds, in particularfrom chemical or combinatorial libraries (i.e., libraries of compoundsthat differ in sequence or size but that have the similar buildingblocks) or by rational, directed or random drug design. See for example,Maulik et al., supra.

In a molecular diversity strategy, large compound libraries aresynthesized, for example, from peptides, oligonucleotides, carbohydratesand/or synthetic organic molecules, using biological, enzymatic and/orchemical approaches. The critical parameters in developing a moleculardiversity strategy include subunit diversity, molecular size, andlibrary diversity. The general goal of screening such libraries is toutilize sequential application of combinatorial selection to obtainhigh-affinity ligands for a desired target, and then to optimize thelead molecules by either random or directed design strategies. Methodsof molecular diversity are described in detail in Maulik, et al., ibid.

Maulik et al. also disclose, for example, methods of directed design, inwhich the user directs the process of creating novel molecules from afragment library of appropriately selected fragments; random design, inwhich the user uses a genetic or other algorithm to randomly mutatefragments and their combinations while simultaneously applying aselection criterion to evaluate the fitness of candidate ligands; and agrid-based approach in which the user calculates the interaction energybetween three dimensional receptor structures and small fragment probes,followed by linking together of favorable probe sites.

According to the present invention, acetolactate synthases can bederived from any Thraustochytriales microorganism, and particularly,from any Schizochytrium microorganism. In one embodiment, a preferredacetolactate synthase of the present invention has an amino acidsequence selected from the group of SEQ ID NO:15, SEQ ID NO:19, SEQ IDNO:22, SEQ ID NO:24. The protein having an amino acid sequencerepresented by SEQ ID NO:15 is a naturally occurring (i.e., wild type)acetolactate synthase from a Thraustochytriales microorganism, andspecifically, is a Schizochytrium acetolactate synthase. The amino acidsequences represented by SEQ ID NO:19, SEQ ID NO:22, SEQ ID NO:24 aresequences that have been modified, such that the resulting enzymes havereduced sensitivity to sulfonylurea compounds, as well as toimidazolinone-class inhibitors and pyrimidinyl oxybenzoates, as comparedto the naturally occurring protein represented by amino acid sequenceSEQ ID NO:15. It is noted that the proteins represented by SEQ ID NO:19,SEQ ID NO:22 and SEQ ID NO:24 have acetolactate synthase biologicalactivity. Acetolactate synthases with reduced sensitivity tosulfonylurea compounds, as well as to imidazolinone-class inhibitors andpyrimidinyl oxybenzoates are preferred acetolactate synthases of thepresent invention, because the nucleic acid sequences encoding suchsynthases can be used in recombinant vectors of the present invention asselectable markers.

Therefore, one embodiment of the present invention relates to a modifiedacetolactate synthase, including any homologue of any of SEQ ID NO:15,SEQ ID NO:19, SEQ ID NO:22 and SEQ ID NO:24, wherein the homologue hasacetolactate synthase biological activity, and particularly, wherein thehomologue has reduced sensitivity to sulfonylurea compounds, as well asto imidazolinone-class inhibitors and pyrimidinyl oxybenzoates, ascompared to the naturally occurring protein represented by amino acidsequence SEQ ID NO:15. In one aspect, such acetolactate synthasehomologues include proteins having an amino acid sequence that differsfrom SEQ ID NO:15 by an amino acid deletion, insertion, or substitutionat one or more of the following positions: 116G, 117A, 192P, 200A, 251K,358M, 383D, 592V, 595W, or 599F. These positions correspond to known ALSmutation sites in a yeast acetolactate synthase (i.e., 116G, 117A, 192P,200A, 251K, 354M, 379D, 583V, 586W, and 590F, respectively) (See Mazurand Falco, 1989, Annu. Rev. Plant Physiol. Plant Mol. Biol. 40:441-470,incorporated herein by reference in its entirety). Other possiblemutation sites will be known to those in the art based on successfulamino acid mutations in ALS from other organisms. The application ofsuch sites to the corresponding sites in the Thraustochytriales ALS isencompassed by the present invention.

As discussed above, the present invention is based in part on thediscovery and production of recombinant constructs for thetransformation of Thraustochytrid microorganisms. Therefore, oneembodiment of the present invention relates to an isolated nucleic acidmolecule comprising a nucleic acid sequence that encodes aThraustochytriales acetolactate synthase, and nucleic acid sequencefully complementary thereto. A nucleic acid molecule encoding anacetolactate synthase of the present invention includes a nucleic acidmolecule encoding any of the acetolactate synthase proteins, includinghomologues, discussed above. More particularly, one embodiment of thepresent invention relates to an isolated nucleic acid moleculecomprising a nucleic acid sequence encoding a protein having an aminoacid sequence that is at least about 65% identical to an amino acidsequence of selected from the group of SEQ ID NO:15, SEQ ID NO:19, SEQID NO:22, SEQ ID NO:24, over at least about 600 amino acids of any ofsuch sequences, wherein the protein is an acetolactate synthase (i.e.,has acetolactate synthase biological activity). More preferably, anisolated nucleic acid molecule of the present invention has a nucleicacid sequence encoding an amino acid sequence that is at least about 70%identical, and more preferably, at least about 75% identical, and evenmore preferably at least about 80% identical, and even more preferablyat least about 85% identical, and even more preferably at least about90% identical and even more preferably at least about 95% identical, andeven more preferably at least about 96% identical, and even morepreferably at least about 97% identical, and even more preferably atleast about 98% identical, and even more preferably at least about 99%identical to any of SEQ ID NO:15, SEQ ID NO:19, SEQ ID NO:22 or SEQ IDNO:24, over at least about 600 amino acids of any of SEQ ID NO:15, SEQID NO:19, SEQ ID NO:22 or SEQ ID NO:24, wherein the protein hasacetolactate synthase biological activity.

In another embodiment, an isolated nucleic acid molecule of the presentinvention has a nucleic acid sequence encoding an amino acid sequencethat is at least about 75% identical to an amino acid sequence ofselected from the group of SEQ ID NO:15, SEQ ID NO:19, SEQ ID NO:22, SEQID NO:24, over at least 95 amino acids of any of such sequences, whereinthe protein is an acetolactate synthase (i.e., has acetolactate synthasebiological activity). More preferably, an isolated nucleic acid moleculeof the present invention has a nucleic acid sequence encoding an aminoacid sequence that is at least about 80% identical, and even morepreferably at least about 85% identical, and even more preferably atleast about 90% identical and even more preferably at least about 95%identical, and even more preferably at least about 96% identical, andeven more preferably at least about 97% identical, and even morepreferably at least about 98% identical, and even more preferably atleast about 99% identical to any of SEQ ID NO:15, SEQ ID NO:19, SEQ IDNO:22 or SEQ ID NO:24, over at least 95 amino acids of any of SEQ IDNO:15, SEQ ID NO:19, SEQ ID NO:22 or SEQ ID NO:24, wherein the proteinhas acetolactate synthase biological activity.

In yet another embodiment, an isolated nucleic acid molecule of thepresent invention has a nucleic acid sequence encoding an amino acidsequence that has any of the above-referenced percent identities to anyof SEQ ID NO:15, SEQ ID NO:19, SEQ ID NO:22 or SEQ ID NO:24 over atleast 100 amino acids, and more preferably 125, and more preferably 150,and more preferably 175, and more preferably 200, and more preferably225, and more preferably 250, and more preferably 275, and morepreferably 300, and more preferably 325, and more preferably 350, andmore preferably 375, and more preferably 400, and more preferably 425,and more preferably 450, and more preferably 475, and more preferably500, and more preferably 525, and more preferably 550, and morepreferably 575 amino acids of any of SEQ ID NO:15, SEQ ID NO:19, SEQ IDNO:22 or SEQ ID NO:24, wherein the protein has acetolactate synthasebiological activity. Percent identity is determined using BLAST 2.0Basic BLAST default parameters, as described above.

In one embodiment, nucleic acid molecules encoding an acetolactatesynthase of the present invention include isolated nucleic acidmolecules that hybridize under moderate stringency conditions, and evenmore preferably under high stringency conditions, and even morepreferably under very high stringency conditions with the complement ofa nucleic acid sequence encoding a naturally occurring acetolactatesynthase. Preferably, an isolated nucleic acid molecule encoding anacetolactate synthase of the present invention comprises a nucleic acidsequence that hybridizes under moderate or high stringency conditions tothe complement of a nucleic acid sequence that encodes a proteincomprising an amino acid sequence represented by SEQ ID NO:15, SEQ IDNO:19, SEQ ID NO:22 or SEQ ID NO:24. In one embodiment, an isolatednucleic acid molecule comprises a nucleic acid sequence that hybridizesunder moderate, high or very high stringency conditions to thecomplement of a nucleic acid sequence represented by nucleotides1260-3314 of SEQ ID NO:14, nucleotides 1260-3314 of SEQ ID NO:18,nucleotides 1260-3314 of SEQ ID NO:21, or nucleotides 1260-3314 of SEQID NO:23.

As used herein, hybridization conditions refer to standard hybridizationconditions under which nucleic acid molecules are used to identifysimilar nucleic acid molecules. Such standard conditions are disclosed,for example, in Sambrook et al., Molecular Cloning: A Laboratory Manual,Cold Spring Harbor Labs Press, 1989. Sambrook et al., ibid., isincorporated by reference herein in its entirety (see specifically,pages 9.31-9.62). In addition, formulae to calculate the appropriatehybridization and wash conditions to achieve hybridization permittingvarying degrees of mismatch of nucleotides are disclosed, for example,in Meinkoth et al., 1984, Anal. Biochem. 138, 267-284; Meinkoth et al.,ibid., is incorporated by reference herein in its entirety.

More particularly, moderate stringency hybridization and washingconditions, as referred to herein, refer to conditions which permitisolation of nucleic acid molecules having at least about 70% nucleicacid sequence identity with the nucleic acid molecule being used toprobe in the hybridization reaction (i.e., conditions permitting about30% or less mismatch of nucleotides). High stringency hybridization andwashing conditions, as referred to herein, refer to conditions whichpermit isolation of nucleic acid molecules having at least about 80%nucleic acid sequence identity with the nucleic acid molecule being usedto probe in the hybridization reaction (i.e., conditions permittingabout 20% or less mismatch of nucleotides). Very high stringencyhybridization and washing conditions, as referred to herein, refer toconditions which permit isolation of nucleic acid molecules having atleast about 90% nucleic acid sequence identity with the nucleic acidmolecule being used to probe in the hybridization reaction (i.e.,conditions permitting about 10% or less mismatch of nucleotides). Asdiscussed above, one of skill in the art can use the formulae inMeinkoth et al., ibid. to calculate the appropriate hybridization andwash conditions to achieve these particular levels of nucleotidemismatch. Such conditions will vary, depending on whether DNA:RNA orDNA:DNA hybrids are being formed. Calculated melting temperatures forDNA:DNA hybrids are 10° C. less than for DNA:RNA hybrids. In particularembodiments, stringent hybridization conditions for DNA:DNA hybridsinclude hybridization at an ionic strength of 6×SSC (0.9 M Na⁺) at atemperature of between about 20° C. and about 35° C. (lower stringency),more preferably, between about 28° C. and about 40° C. (more stringent),and even more preferably, between about 35° C. and about 45° C. (evenmore stringent), with appropriate wash conditions. In particularembodiments, stringent hybridization conditions for DNA:RNA hybridsinclude hybridization at an ionic strength of 6×SSC (0.9 M Na⁺) at atemperature of between about 30° C. and about 45° C., more preferably,between about 38° C. and about 50° C., and even more preferably, betweenabout 45° C. and about 55° C., with similarly stringent wash conditions.These values are based on calculations of a melting temperature formolecules larger than about 100 nucleotides, 0% formamide and a G+Ccontent of about 40%. Alternatively, T_(m) can be calculated empiricallyas set forth in Sambrook et al., supra, pages 9.31 to 9.62. In general,the wash conditions should be as stringent as possible, and should beappropriate for the chosen hybridization conditions. For example,hybridization conditions can include a combination of salt andtemperature conditions that are approximately 20-25° C. below thecalculated T_(m) of a particular hybrid, and wash conditions typicallyinclude a combination of salt and temperature conditions that areapproximately 12-20° C. below the calculated T_(m) of the particularhybrid. One example of hybridization conditions suitable for use withDNA:DNA hybrids includes a 2-24 hour hybridization in 6×SSC (50%formamide) at about 42° C., followed by washing steps that include oneor more washes at room temperature in about 2×SSC, followed byadditional washes at higher temperatures and lower ionic strength (e.g.,at least one wash as about 37° C. in about 0.1×-0.5×SSC, followed by atleast one wash at about 68° C. in about 0.1×-0.5×SSC).

In another embodiment, nucleic acid molecules encoding an acetolactatesynthase of the present invention include isolated nucleic acidmolecules comprising a nucleic acid sequence encoding a protein havingan amino acid sequence comprising at least 30 contiguous amino acidresidues of any of SEQ ID NO:15, SEQ ID NO:19, SEQ ID NO:22 or SEQ IDNO:24, (i.e., 30 contiguous amino acid residues having 100% identitywith 30 contiguous amino acids of any of SEQ ID NO:15, SEQ ID NO:19, SEQID NO:22 or SEQ ID NO:24). In a preferred embodiment, an isolatednucleic acid molecule comprises a nucleic acid sequence encoding aprotein having an amino acid sequence comprising at least 50, and morepreferably at least 75, and more preferably at least 100, and morepreferably at least 115, and more preferably at least 130, and morepreferably at least 150, and more preferably at least 200, and morepreferably, at least 250, and more preferably, at least 300, and morepreferably, at least 350, and more preferably, at least 400, and morepreferably, at least 450, and more preferably, at least 500, and morepreferably, at least 550, and more preferably, at least 600, and morepreferably, at least 650, contiguous amino acid residues of any of SEQID NO:15, SEQ ID NO:19, SEQ ID NO:22 or SEQ ID NO:24. Such a protein hasacetolactate synthase biological activity. In one embodiment, anisolated nucleic acid molecule encoding an acetolactate synthasecomprises a nucleic acid sequence having at least 60 contiguousnucleotides, and more preferably at least 150, and more preferably atleast 225, and more preferably at least 300, and more preferably atleast 345, and more preferably at least 390, and more preferably atleast 450, and more preferably at least 525, and more preferably atleast 600, and more preferably at least 750, and more preferably atleast 900, and more preferably at least 1050, and more preferably atleast 1200, and more preferably at least 1350, and more preferably atleast 1500, and more preferably at least 1650, and more preferably atleast 1800, and even more preferably at least 1950, contiguousnucleotides of nucleotides 1260-3314 of SEQ ID NO:15, nucleotides1260-3314 of SEQ ID NO:18, nucleotides 1260-3314 of SEQ ID NO:21, ornucleotides 1260-3314 of SEQ ID NO:23.

Particularly preferred nucleic acid molecules of the present inventioninclude nucleotides 1260-3314 of SEQ ID NO:14 (encodes SEQ ID NO:15),nucleotides 1260-3314 of SEQ ID NO:18 (encodes SEQ ID NO:19),nucleotides 1260-3314 of SEQ ID NO:21 (encodes SEQ ID NO:22), ornucleotides 1260-3314 of SEQ ID NO:23 (encodes SEQ ID NO:24), SEQ IDNO:14, SEQ ID NO:18, SEQ ID NO:21 or SEQ ID NO:23.

In accordance with the present invention, an isolated nucleic acidmolecule is a nucleic acid molecule that has been removed from itsnatural milieu (i.e., that has been subject to human manipulation), itsnatural milieu being the genome or chromosome in which the nucleic acidmolecule is found in nature. As such, “isolated” does not necessarilyreflect the extent to which the nucleic acid molecule has been purified,but indicates that the molecule does not include an entire genome or anentire chromosome in which the nucleic acid molecule is found in nature.An isolated nucleic acid molecule can include a gene, such as anacetolactate synthase gene described herein. An isolated nucleic acidmolecule that includes a gene is not a fragment of a chromosome thatincludes such gene, but rather includes the coding region and regulatoryregions associated with the gene, but no additional genes naturallyfound on the same chromosome. An isolated nucleic acid molecule can alsoinclude a specified nucleic acid sequence flanked by (i.e., at the 5′and/or the 3′ end of the sequence) additional nucleic acids that do notnormally flank the specified nucleic acid sequence in nature (i.e., areheterologous sequences). Isolated nucleic acid molecule can include DNA,RNA (e.g., mRNA), or derivatives of either DNA or RNA (e.g., cDNA).Although the phrase “nucleic acid molecule” primarily refers to thephysical nucleic acid molecule and the phrase “nucleic acid sequence”primarily refers to the sequence of nucleotides on the nucleic acidmolecule, the two phrases can be used interchangeably, especially withrespect to a nucleic acid molecule, or a nucleic acid sequence, beingcapable of encoding a protein.

Preferably, an isolated nucleic acid molecule of the present inventionis produced using recombinant DNA technology (e.g., polymerase chainreaction (PCR) amplification, cloning) or chemical synthesis. Isolatednucleic acid molecules include natural nucleic acid molecules andhomologues thereof, including, but not limited to, natural allelicvariants and modified nucleic acid molecules in which nucleotides havebeen inserted, deleted, substituted, and/or inverted in such a mannerthat such modifications provide the desired effect on protein biologicalactivity. Allelic variants and protein homologues (e.g., proteinsencoded by nucleic acid homologues) have been discussed in detail above.

A nucleic acid molecule homologue can be produced using a number ofmethods known to those skilled in the art (see, for example, Sambrook etal., ibid.). For example, nucleic acid molecules can be modified using avariety of techniques including, but not limited to, classic mutagenesistechniques and recombinant DNA techniques, such as site-directedmutagenesis, chemical treatment of a nucleic acid molecule to inducemutations, restriction enzyme cleavage of a nucleic acid fragment,ligation of nucleic acid fragments, PCR amplification and/or mutagenesisof selected regions of a nucleic acid sequence, synthesis ofoligonucleotide mixtures and ligation of mixture groups to “build” amixture of nucleic acid molecules and combinations thereof. Nucleic acidmolecule homologues can be selected from a mixture of modified nucleicacids by screening for the function of the protein encoded by thenucleic acid and/or by hybridization with a wild-type gene.

Similarly, the minimum size of a nucleic acid molecule of the presentinvention is a size sufficient to encode a protein having the desiredbiological activity, or sufficient to form a probe or oligonucleotideprimer that is capable of forming a stable hybrid with the complementarysequence of a nucleic acid molecule encoding the natural protein (e.g.,under moderate, high or very high stringency conditions). As such, thesize of the nucleic acid molecule encoding such a protein can bedependent on nucleic acid composition and percent homology or identitybetween the nucleic acid molecule and complementary sequence as well asupon hybridization conditions per se (e.g., temperature, saltconcentration, and formamide concentration). The minimal size of anucleic acid molecule that is used as an oligonucleotide primer or as aprobe is typically at least about 12 to about 15 nucleotides in lengthif the nucleic acid molecules are GC-rich and at least about 15 to about18 bases in length if they are AT-rich. There is no limit, other than apractical limit, on the maximal size of a nucleic acid molecule of thepresent invention, in that the nucleic acid molecule can include aportion of a protein-encoding sequence (e.g., an acetolactatesynthase-encoding sequence) or a nucleic acid sequence encoding afull-length protein.

One embodiment of the present invention includes a recombinant vector tobe used for transformation of a Thraustochytriales microorganism.According to the present invention, a recombinant vector is anengineered (i.e., artificially produced) nucleic acid molecule that isused as a tool for manipulating a nucleic acid sequence of choice andfor introducing such a nucleic acid sequence into a host cell. Therecombinant vector is therefore suitable for use in cloning, sequencing,and/or otherwise manipulating the nucleic acid sequence of choice, suchas by expressing and/or delivering the nucleic acid sequence of choiceinto a host cell to form a recombinant cell. Such a vector typicallycontains heterologous nucleic acid sequences, that is nucleic acidsequences that are not naturally found adjacent to nucleic acid sequenceto be delivered, although the vector can also contain regulatory nucleicacid sequences (e.g., promoters, untranslated regions) which arenaturally found adjacent to nucleic acid molecules of the presentinvention (discussed in detail below). The vector can be either RNA orDNA, either prokaryotic or eukaryotic, and typically is a plasmid. Thevector can be maintained as an extrachromosomal element (e.g., aplasmid) or it can be integrated into the chromosome of the recombinantmicroorganism. The entire vector can remain in place within a host cell,or under certain conditions, the plasmid DNA can be deleted, leavingbehind the nucleic acid molecule of the present invention. Theintegrated nucleic acid molecule can be under chromosomal promotercontrol, under native or plasmid promoter control, or under acombination of several promoter controls. Single or multiple copies ofthe nucleic acid molecule can be integrated into the chromosome. Arecombinant vector of the present invention contains at least oneselectable marker for Thraustochytriales microorganisms according to thepresent invention, such as a nucleic acid sequence encoding aThraustochytriales acetolactate synthase (natural protein or homologue)or a nucleic acid sequence encoding the ble gene (described below). Asused herein, the phrase “recombinant nucleic acid molecule” is usedprimarily to refer to a recombinant vector into which has been ligatedthe nucleic acid sequence to be cloned, manipulated, transformed intothe host cell (i.e., the insert).

Typically, a recombinant vector, and therefore a recombinant nucleicacid molecule, includes at least one nucleic acid molecule of thepresent invention operatively linked to one or more transcriptioncontrol sequences. As used herein, the phrase “recombinant molecule” or“recombinant nucleic acid molecule” primarily refers to a nucleic acidmolecule or nucleic acid sequence operatively linked to a transcriptioncontrol sequence, but can be used interchangeably with the phrase“nucleic acid molecule”, when such nucleic acid molecule is arecombinant molecule as discussed herein. According to the presentinvention, the phrase “operatively linked” refers to linking a nucleicacid molecule to a transcription control sequence in a manner such thatthe molecule is able to be expressed when transfected (i.e.,transformed, transduced, transfected, conjugated or conduced) into ahost cell. Transcription control sequences are sequences which controlthe initiation, elongation, or termination of transcription.Particularly important transcription control sequences are those whichcontrol transcription initiation, such as promoter, enhancer, operatorand repressor sequences. Suitable transcription control sequencesinclude any transcription control sequence that can function in amicroorganism of the order Thraustochytriales. The present inventors arebelieved to be the first to isolate and identify at least three of suchpromoters, described in detail elsewhere herein.

Preferred promoters include, but are not limited to, aThraustochytriales acetolactate synthase promoter (represented herein bynucleotides 1-1259 of SEQ ID NO:14), a Thraustochytriales α-tubulinpromoter (represented herein by nucleotides 441-894 of SEQ ID NO:9, apromoter from a Thraustochytriales polyketide synthase (PKS) system(contained within SEQ ID NO:34), and a Thraustochytriales fatty aciddesaturase promoter (contained within SEQ ID NO:31; it is noted that thefatty acid desaturase promoter is referred to as a desaturase promoterin U.S. Provisional Application Ser. No. 60/284,116, supra.). Thecloning and sequencing of the α-tubulin promoter, the acetolactatesynthase promoter, and the fatty acid desaturase promoter are describedin the Examples section. In a preferred embodiment, the α-tubulinpromoter comprises the naturally occurring Thraustochytriales α-tubulinpromoter sequence (nucleotides 441-894 of SEQ ID NO:9), or a nucleicacid sequence that is at least 95% identical to nucleotides 441-894 ofSEQ ID NO:9, wherein the promoter has at least basal α-tubulin promotertranscriptional activity. Similarly, a preferred acetolactate synthasepromoter comprises a nucleic acid sequence of the naturally occurringThraustochytriales acetolactate synthase promoter (represented withinnucleotides 1-1259 of SEQ ID NO:14), or a nucleic acid sequence that isat least 75%, and more preferably 80%, and more preferably 85%, and morepreferably 90%, and more preferably 95% identical to nucleotides 1-1259of SEQ ID NO:14, wherein the promoter as at least basal acetolactatesynthase promoter transcriptional activity. A preferred PKS promotercomprises a nucleic acid sequence of a naturally occurringThraustochytriales PKS promoter (represented within SEQ ID NO:34), or anucleic acid sequence that is at least 75%, and more preferably 80%, andmore preferably 85%, and more preferably 90%, and more preferably 95%identical to SEQ ID NO:34, wherein the promoter as at least basal PKSpromoter transcriptional activity. Finally, a preferred fatty aciddesaturase promoter comprises a nucleic acid sequence of the naturallyoccurring Thraustochytriales fatty acid desaturase promoter (representedwithin SEQ ID NO:31), or is contained within a nucleic acid sequencethat is at least 75%, and more preferably 80%, and more preferably 85%,and more preferably 90%, and more preferably 95% identical to SEQ IDNO:31, wherein the promoter as at least basal fatty acid desaturasepromoter transcriptional activity. Methods for determining percentidentity have been previously described herein for the acetolactatesynthase sequences, and are encompassed herein.

In one embodiment, a recombinant vector of the present invention is anexpression vector. As used herein, the phrase “expression vector” isused to refer to a vector that is suitable for production of an encodedproduct (e.g., a protein of interest). In this embodiment, a nucleicacid sequence encoding the product to be produced is inserted into therecombinant vector to produce a recombinant nucleic acid molecule. Thenucleic acid sequence encoding the protein to be produced is insertedinto the vector in a manner that operatively links the nucleic acidsequence to regulatory sequences in the vector (e.g., aThraustochytriales promoter of the present invention) which enable thetranscription and translation of the nucleic acid sequence within therecombinant microorganism. The selectable markers of the presentinvention enable the selection of a recombinant microorganism into whicha recombinant nucleic acid molecule of the present invention hassuccessfully been introduced.

In another embodiment, a recombinant vector of the present invention isa targeting vector. As used herein, the phrase “targeting vector” isused to refer to a vector that is used to deliver a particular nucleicacid molecule into a recombinant cell, wherein the nucleic acid moleculeis used to delete or inactivate an endogenous gene within the host cell(i.e., used for targeted gene disruption or knock-out technology). Sucha vector may also be known in the art as a “knock-out” vector. In oneaspect of this embodiment, a portion of the vector, but more typically,the nucleic acid molecule inserted into the vector (i.e., the insert),has a nucleic acid sequence that is homologous to a nucleic acidsequence of a target gene in the host cell (i.e., a gene which istargeted to be deleted or inactivated). The nucleic acid sequence of thevector insert is designed to bind to the target gene such that thetarget gene and the insert undergo homologous recombination, whereby theendogenous target gene is deleted, inactivated or attenuated (i.e., byat least a portion of the endogenous target gene being mutated ordeleted).

In one embodiment, a preferred recombinant vector of the presentinvention is a recombinant vector that is suitable for use in aThraustochytriales microorganism, and which includes a nucleic acidsequence encoding an acetolactate synthase molecule of the presentinvention. Preferably, the acetolactate synthase is modified as comparedto the naturally occurring form (SEQ ID NO:15), such that the synthaseconfers onto a Thraustochytriales microorganism that has beentransfected with the recombinant vector, a reduced sensitivity tosulfonurea compounds, imidazolinone-class inhibitors, and/or pyrimidinyloxybenzoates. Preferably, such a recombinant vector comprises a nucleicacid sequence encoding an acetolactate synthase protein comprising anamino acid sequence that differs from SEQ ID NO:15 by an amino aciddeletion, insertion, or substitution at one or more of the followingpositions: 116G, 117A, 192P, 200A, 251K, 358M, 383D, 592V, 595W, or599F. In one embodiment, such acetolactate synthase proteins have anamino acid sequence including, but not limited to: SEQ ID NO:19, SEQ IDNO:22 and SEQ ID NO:24. Preferably, such a recombinant vector comprisesa nucleic acid sequence selected from: nucleotides 1260-3314 of SEQ IDNO:18, nucleotides 1260-3314 of SEQ ID NO:21, and nucleotides 1260-3314of SEQ ID NO:23. In a particularly preferred embodiment, recombinantvectors containing ALS-encoding nucleic acid sequences that confer thedesired resistance include SEQ ID NO:18, SEQ ID NO:21 and SEQ ID NO:23.

In one embodiment, a recombinant vector that is suitable for conferringonto a Thraustochytriales microorganism that has been transfected withthe recombinant vector a reduced sensitivity to sulfonurea compounds,imidazolinone-class inhibitors, and/or pyrimidinyl oxybenzoates,comprises SEQ ID NO:15, which is the naturally occurring Schizochytriumacetolactate synthase sequence. In this embodiment, the recombinantvector is designed to overexpress the naturally occurring synthase,whereby such overexpression has the effect of conferring resistance tothe specified compounds onto the microorganism. In this embodiment, itwill be appreciated by one skilled in the art that use of recombinantDNA technologies can improve control of expression of transformednucleic acid molecules by manipulating, for example, the number ofcopies of the nucleic acid molecules within the host cell, theefficiency with which those nucleic acid molecules are transcribed, theefficiency with which the resultant transcripts are translated, and theefficiency of post-translational modifications. Additionally, thepromoter sequence might be genetically engineered to improve the levelof expression as compared to the native promoter. Recombinant techniquesuseful for controlling the expression of nucleic acid molecules include,but are not limited to, integration of the nucleic acid molecules intoone or more host cell chromosomes, addition of vector stabilitysequences to plasmids, substitutions or modifications of transcriptioncontrol signals (e.g., promoters, operators, enhancers), substitutionsor modifications of translational control signals (e.g., ribosomebinding sites, Shine-Dalgarno sequences), modification of nucleic acidmolecules to correspond to the codon usage of the host cell, anddeletion of sequences that destabilize transcripts.

In one embodiment of the present invention, a recombinant vectorsuitable for use in the transformation of Thraustochytrialesmicroorganisms contains the Sh ble gene from Streptoalloteichushindustanus as a selectable marker (which encodes a “bleomycin-bindingprotein), in combination with a Thraustochytriales promoter aspreviously described herein. A preferred recombinant vector comprisingthe ble gene and a Thraustochytriales promoter includes, for example,the vector sequence represented by SEQ ID NO:8 or 9. The amino acidsequence of the Streptoalloteichus hindustanus bleomycin binding proteinis represented herein as SEQ ID NO:10.

Recombinant nucleic acid molecules of the present invention, which canbe either DNA or RNA, can also contain additional regulatory sequences,such as translation regulatory sequences, origins of replication, andother regulatory sequences that are compatible with the recombinantcell. In one embodiment, a recombinant molecule of the presentinvention, including those which are integrated into the host cellchromosome, also contains secretory signals (i.e., signal segmentnucleic acid sequences) to enable an expressed protein to be secretedfrom the cell that produces the protein. Suitable signal segmentsinclude a signal segment that is naturally associated with the proteinto be expressed or any heterologous signal segment capable of directingthe secretion of the protein according to the present invention. Inanother embodiment, a recombinant molecule of the present inventioncomprises a leader sequence to enable an expressed protein to bedelivered to and inserted into the membrane of a host cell. Suitableleader sequences include a leader sequence that is naturally associatedwith the protein, or any heterologous leader sequence capable ofdirecting the delivery and insertion of the protein to the membrane of acell.

Having described various tools which are useful for transformingmicroorganisms of the order Thraustochytriales, one embodiment of thepresent invention relates to a method for transformation of cells of amicroorganism of the Order Thraustochytriales. The method includes afirst step of: (a) introducing into cells of a microorganism of theOrder Thraustochytriales a recombinant nucleic acid molecule asdescribed previously herein. The recombinant nucleic acid moleculecomprises a nucleic acid sequence encoding an acetolactate synthase thatconfers onto said cells reduced sensitivity to compounds selected fromthe group consisting of: sulfonylurea compounds, imidazolinone-classinhibitors, and pyrimidinyl oxybenzoates. Such acetolactate synthaseshave been described in detail above and include acetolactate synthaseshaving an amino acid sequence represented by SEQ ID NO:19, SEQ ID NO:22and SEQ ID NO:24, as well as homologues of any of such sequences or SEQID NO:15 as discussed above. The method includes a second step of: (b)selecting cells that have been successfully transformed with therecombinant nucleic acid molecule by culturing the cells of (a) in amedium containing at least one compound that is inhibitory tountransformed cells, the compound being selected from the groupconsisting of: a sulfonylurea compound, an imidazolinone-classinhibitor, and pyrimidinyl oxybenzoates. Cells which grow in thepresence of such compounds have been successfully transformed.

The recombinant nucleic acid molecule used in the present methodcomprises any of the recombinant vectors of the present inventionpreviously described herein, and typically includes at least one nucleicacid sequence encoding a protein to be produced by the recombinant cell(i.e., comprising a recombinant expression vector), or a nucleic acidsequence useful for targeted deletion or inactivation of an endogenousgene in the recombinant cell (i.e., comprising a recombinant targetingvector).

In one embodiment, the recombinant nucleic acid molecule furthercomprises a nucleic acid sequence encoding a protein to be produced bythe cell, wherein the nucleic acid sequence encoding the protein isoperatively linked to a transcription control sequence. Proteins thatmay be desirable for production in a Thraustochytriales will be known tothose of skill in the art, and all are intended to be encompassed by thepresent invention. Particularly preferred proteins include, but are notlimited to, proteins associated with the synthesis of a fatty acidselected from the group consisting of docosahexaenoic acid (DHA),docosapentaenoic acid (DPA), eicosapentaenoic acid (EPA) and arachadonicacid (ARA). Such proteins include, for example, a fatty acid synthase, afatty acid desaturase, a fatty acid elongase, a protein associated witha polyketide synthase complex and a protein associated withincorporation of fatty acids into phospholipids or into triacylglycerolmolecules. In one aspect, the protein is an ω-3 fatty acid desaturase isencoded by a nucleic acid sequence SEQ ID NO:29. SEQ ID NO:30 representsthe amino acid sequence of the desaturase. In another aspect, theprotein is a polyenoic fatty acid isomerase. In one embodiment, proteinswhich can be produced in Thraustochytriales microorganisms using thepresent method include proteins associated with the isoprenoidbiosynthetic pathways. Such proteins include, but are not limited to,HMG-CoA synthase, HMG-CoA reductase, squalene synthase, phytoenesynthase, phytoene desaturase, a carotenoid cyclase, a carotenoidhyroxylase, a carotenoid ketolase. In yet another embodiment, proteinswhich can be produced in Thraustochytriales microorganisms using thepresent method include, but are not limited to, vitamin E and lipoicacid.

In one embodiment, the recombinant nucleic acid molecule useful in themethod of the present invention includes a nucleic acid sequence thathybridizes with a target nucleic acid sequence in the microorganism suchthat a gene comprising the target nucleic acid sequence is mutated orinactivated by homologous recombination with the second nucleic acidsequence. Such a nucleic acid sequence can be homologous to genes thatencode enzymes (or nucleic acids which regulate the expression of suchgenes) of the saturated and polyunsaturated fatty acid synthesispathways, genes encoding proteins that are involved in the degradationof other valuable compounds produced by the Thraustochytrialesmicroorganism or which otherwise lessen the value of the desiredcompound, or genes encoding proteins that are associated with thesynthesis of compounds whose synthesis is in competition with othermolecules of interest. For example, target nucleic acid sequencesinclude, but are not limited to, sequences encoding lipases, fatty acidoxidation enzymes, proteins involved in carbohydrate synthesis, proteinsinvolved in synthesis of products of isoprenoid pathways, proteinsinvolved in synthesis of cell wall components, proteins involved in thesaturated fatty acid synthesis pathways, proteins involved in thepolyunsaturated fatty acid synthesis pathways, proteins associated witha polyketide synthase complex, and proteins associated withincorporation of fatty acids into phospholipids or triacylglycerolmolecules.

In one embodiment of the present invention, the method fortransformation of Thraustochytriales microorganisms includes a step ofintroducing into the cell at least one additional recombinant nucleicacid molecule comprising a nucleic acid sequence encoding a protein tobe expressed, the nucleic acid sequence being operatively linked to atranscription control sequence. Alternatively, the additionalrecombinant nucleic acid molecule can include a second nucleic acidsequence that hybridizes with a target nucleic acid sequence in themicroorganism such that a gene comprising the target nucleic acidsequence is mutated or inactivated by homologous recombination with thesecond nucleic acid sequence. In this manner, multiple proteins can beintroduced into the cell, multiple genes can be inactivated, orcombinations of the two are possible. The additional recombinant nucleicacid molecule can be introduced into the Thraustochytrialesmicroorganism simultaneously with the first recombinant nucleic acidmolecule (i.e., cotransformation), or as a subsequent transformation(e.g., for the purposes of “stacking” traits).

In one embodiment, the method further includes the step of introducinginto the cell at least one additional recombinant nucleic acid moleculecomprising a nucleic acid sequence encoding a bleomycin-binding protein.In this embodiment, the additional recombinant nucleic acid molecule ispreferably introduced in a subsequent step, rather than as acotransformation. Preferably, the recombinant nucleic acid moleculecomprising a nucleic acid sequence encoding a bleomycin-binding proteinfurther comprises a nucleic acid sequence encoding a second protein tobe expressed by the cell, wherein the nucleic acid sequence encoding thesecond protein is operatively linked to a transcription controlsequence. Alternatively, or in addition, the recombinant nucleic acidmolecule comprising a nucleic acid sequence encoding a bleomycin-bindingprotein further comprises a second nucleic acid sequence that hybridizeswith a target nucleic acid sequence in the microorganism such that agene comprising the target nucleic acid sequence is mutated orinactivated by homologous recombination with the second nucleic acidsequence. In one embodiment, such a recombinant nucleic acid moleculecomprises a nucleic acid sequence SEQ ID NO:9.

Suitable host cells to transform using the method of the presentinvention include, but are not limited to, any microorganism of theorder Thraustochytriales. Host cells can be either untransformed cellsor cells that are already transfected with at least one nucleic acidmolecule. Preferred host cells for use in the present invention includemicroorganisms from a genus including, but not limited to:Thraustochytrium, Labyrinthuloides, Japonochytrium, and Schizochytrium.Preferred species within these genera include, but are not limited to:any Schizochytrium species, including Schizochytrium aggregatum,Schizochytrium limacinum, Schizochytrium minutum; any Thraustochytriumspecies (including former Ulkenia species such as U. visurgensis, U.amoeboida, U. sarkariana, U. profunda, U. radiata, U. minuta and Ulkeniasp. BP-5601), and including Thraustochytrium striatum, Thraustochytriumaureum, Thraustochytrium roseum; and any Japonochytrium species.Particularly preferred strains of Thraustochytriales include, but arenot limited to: Schizochytrium sp. (S31) (ATCC 20888); Schizochytriumsp. (S8) (ATCC 20889); Schizochytrium sp. (LC-RM) (ATCC 18915);Schizochytrium sp. (SR21); Schizochytrium aggregatum (Goldstein etBelsky) (ATCC 28209); Schizochytrium limacinum (Honda et Yokochi) (IFO32693); Thraustochytrium sp. (23B) (ATCC 20891); Thraustochytriumstriatum (Schneider) (ATCC 24473); Thraustochytrium aureum (Goldstein)(ATCC 34304); Thraustochytrium roseum (Goldstein) (ATCC 28210); andJaponochytrium sp. (L1) (ATCC 28207).

According to the present invention, the term “transformation” is used torefer to any method by which an exogenous nucleic acid molecule (i.e., arecombinant nucleic acid molecule) can be inserted into microbial cells,such Thraustochytriales microbial cells. In microbial systems, the term“transformation” is used to describe an inherited change due to theacquisition of exogenous nucleic acids by the microorganism and isessentially synonymous with the term “transfection”. Suitabletransformation techniques include, but are not limited to, particlebombardment, electroporation, microinjection, lipofection, adsorption,infection and protoplast fusion.

In one embodiment, a protein to be produced using a method of thepresent invention is produced by culturing a cell that expresses theprotein (i.e., a recombinant Thraustochytriales microorganism) underconditions effective to produce the protein. In some instances, theprotein may be recovered, and in others, the microorganism may beharvested in whole or as a lysate and used as a “biomass”. In anotherembodiment, a target gene is deleted or inactivated by culturing a cellthat has been transformed with a recombinant molecule comprising atargeting vector of the present invention under conditions effective toallow recombination within the cell, resulting in deletion orinactivation of a target gene. A preferred cell to culture is arecombinant cell of the present invention. Effective culture conditionsinclude, but are not limited to, effective media, bioreactor,temperature, pH and oxygen conditions that permit protein productionand/or recombination. An effective medium refers to any medium in whicha Thraustochytriales cell is typically cultured. Such medium typicallycomprises an aqueous medium having assimilable carbon, nitrogen andphosphate sources, and appropriate salts, minerals, metals and othernutrients, such as vitamins. Examples of suitable media and cultureconditions are discussed in detail in the Examples section. Cultureconditions suitable for Thraustochytriales microorganisms are alsodescribed in U.S. Pat. No. 5,340,742, issued Aug. 23, 1994, to Barclay;incorporated herein by reference in its entirety. Cells of the presentinvention can be cultured in conventional fermentation bioreactors,shake flasks, test tubes, microtiter dishes, and petri plates. Culturingcan be carried out at a temperature, pH and oxygen content appropriatefor a recombinant cell. Such culturing conditions are within theexpertise of one of ordinary skill in the art.

Depending on the vector and host system used for production, resultantproteins of the present invention may either remain within therecombinant cell; be secreted into the fermentation medium; be secretedinto a space between two cellular membranes; or be retained on the outersurface of a cell membrane. The phrase “recovering the protein” refersto collecting the whole fermentation medium containing the protein andneed not imply additional steps of separation or purification. Proteinsproduced by the method of the present invention can be purified using avariety of standard protein purification techniques, such as, but notlimited to, affinity chromatography, ion exchange chromatography,filtration, electrophoresis, hydrophobic interaction chromatography, gelfiltration chromatography, reverse phase chromatography, concanavalin Achromatography, chromatofocusing and differential solubilization.Proteins produced by the method of the present invention are preferablyretrieved in “substantially pure” form. As used herein, “substantiallypure” refers to a purity that allows for the effective use of theprotein as a commercial product.

Yet another embodiment of the present invention relates to a recombinantmicroorganism of the order Thraustochytriales, which has beentransformed with a recombinant nucleic acid molecule comprising anucleic acid sequence encoding an acetolactate synthase of the presentinvention. Preferably, the acetolactate synthase confers onto themicroorganism reduced sensitivity to compounds selected from the groupconsisting of: sulfonylurea compounds, imidazolinone-class inhibitors,and pyrimidinyl oxybenzoates. Suitable recombinant nucleic acidmolecules and sequences for use in transforming such a microorganismhave been described in detail above. Such a microorganism can be furthertransformed with other recombinant nucleic acid molecules, includingrecombinant nucleic acid molecules comprising a ble gene selectablemarker and Thraustochytriales transcription control sequences, aspreviously described herein. Recombinant Thraustochytrialesmicroorganisms according to the present invention are described in theExamples section. According to the present invention, a recombinantThraustochytriales microorganism of the present invention is geneticallyengineered to express a protein of interest (examples of such proteinsare discussed above) using the recombinant vectors described herein,and/or is genetically engineered for a targeted deletion or inactivationof a target gene using the recombinant vectors described herein.

As used herein, a recombinant microorganism has a genome which ismodified (i.e., mutated or changed) from its normal (i.e., wild-type ornaturally occurring) form using recombinant technology. A recombinantmicroorganism according to the present invention can include amicroorganism in which nucleic acid molecules have been inserted,deleted or modified (i.e., mutated; e.g., by insertion, deletion,substitution, and/or inversion of nucleotides), in such a manner thatsuch modifications provide the desired effect within the microorganism.As used herein, genetic modifications which result in a decrease in geneexpression, in the function of the gene, or in the function of the geneproduct (i.e., the protein encoded by the gene) can be referred to asinactivation (complete or partial), deletion, interruption, blockage ordown-regulation of a gene. For example, a genetic modification in a genewhich results in a decrease in the function of the protein encoded bysuch gene, can be the result of a complete deletion of the gene (i.e.,the gene does not exist, and therefore the protein does not exist), amutation in the gene which results in incomplete or no translation ofthe protein (e.g., the protein is not expressed), or a mutation in thegene which decreases or abolishes the natural function of the protein(e.g., a protein is expressed which has decreased or no enzymaticactivity or action). Genetic modifications which result in an increasein gene expression or function can be referred to as amplification,overproduction, overexpression, activation, enhancement, addition, orup-regulation of a gene.

According to the present invention, a recombinant Thraustochytrialesmicroorganism can be produced using any microorganism of the orderThraustochytriales. Preferred genera of Thraustochytriales include, butare not limited to: Thraustochytrium, Labyrinthuloides, Japonochytrium,and Schizochytrium. Preferred species within these genera include, butare not limited to: any Schizochytrium species, including Schizochytriumaggregatum, Schizochytrium limacinum; any Thraustochytrium species(including any former Ulkenia species such as U. visurgensis, U.amoeboida, U. sarkariana, U. profunda, U. radiata, U. minuta and Ulkeniasp. BP-5601), Thraustochytrium striatum, Thraustochytrium aureum,Thraustochytrium roseum; and any Japonochytrium species. Particularlypreferred strains of Thraustochytriales include, but are not limited to:Schizochytrium sp. (S31) (ATCC 20888); Schizochytrium sp. (S8) (ATCC20889); Schizochytrium sp. (LC-RM) (ATCC 18915); Schizochytrium sp.(SR21); Schizochytrium aggregatum (Goldstein et Belsky) (ATCC 28209);Schizochytrium limacinum (Honda et Yokochi) (IFO 32693);Thraustochytrium sp. (23B) (ATCC 20891); Thraustochytrium striatum(Schneider) (ATCC 24473); Thraustochytrium aureum (Goldstein) (ATCC34304); Thraustochytrium roseum (Goldstein) (ATCC 28210); andJaponochytrium sp. (L1) (ATCC 28207).

The following examples are provided for the purpose of illustration andare not intended to limit the scope of the present invention.

EXAMPLES Example 1

This example describes the production of recombinant plasmidpTUBZEO11-2.

Construction of recombinant plasmid pTUBZEO11-2 is illustrated in FIGS.1 and 2. This plasmid contains the ble gene from Streptoalloteichushindustanus functionally coupled to an α-tubulin gene promoter isolatedfrom Schizochytrium sp. This plasmid was produced as follows. A cDNAclone (CGNE0002-001-B6) was isolated from a Schizochytrium sp. cDNAlibrary and partially sequenced (SEQ ID NO:1) as part of a large-scaleSchizochytrium cDNA sequencing project. The nucleotide sequence wasdetermined to encode α-tubulin by BLASTX homology searching (Gish, W.and D. States. 1993. Nat. Genet. 3:266-272). The amino acid sequencededuced from bases 116 through 550 is 93% identical to the first 145amino acids of α-tubulin from Pelvetica fastigiata (GenBank AccessionNo. U58642).

In order to isolate the promoter associated with this gene, genomic DNAwas isolated from Schizochytrium sp. cells and processed by the use of a“GenomeWalker™” kit (Clontech Laboratories, Inc., Palo Alto, Calif.),which involves enzymatic digestion of genomic DNA with restrictionendonucleases to generate blunt ends, followed by ligation of thedigested DNA to specific double-stranded DNA adapter molecules providedin the kit. The DNA upstream of the α-tubulin coding sequence was thenamplified by the polymerase chain reaction (PCR), using the outeradapter primer (AP1) provided in the kit and the α-tubulin-specificprimer PGR20 (SEQ ID NO:2). Further amplification of the gene wascarried out using the nested adapter primer (AP2) provided in the kitand a nested α-tubulin-specific primer PGR19 (SEQ ID NO:3). Theresulting PCR products were subcloned into plasmid pCR2.1-TOPO(Invitrogen Corp., Carlsbad, Calif.). One of the subcloned fragments wassequenced; the sequence of the 725 bp immediately preceding theα-tubulin gene start codon is given as SEQ ID NO:4.

Using oligonucleotide primers based on the DNA sequence obtained in thismanner, PCR using Taq DNA polymerase (Perkin-Elmer Corp., Norwalk,Conn.) was used to generate a modified α-tubulin promoter region inwhich an NcoI restriction site was incorporated into the 3′ end of theDNA fragment; this NcoI site contained a start codon that was at thesame position as in the α-tubulin coding region. The primers used inthis reaction were PGR33 (SEQ ID NO:5) and PGR34 (SEQ ID NO:6), and thetemplate was genomic DNA isolated from Schizochytrium sp. cells. Thefollowing reaction conditions were utilized: 94° C. for 4 min; (94° C.for 1 min, 54° C. for 45 sec, 72° C. for 2 min)×30; 72° C. for 7 min.This fragment was cloned into plasmid pCR2.1-TOPO to form plasmid p7TUB(SEQ ID NO:7). Plasmid p7TUB was digested with NcoI, and a resulting463-bp fragment containing the Schizochytrium α-tubulin promoter regionwas isolated by agarose gel purification. Plasmid pSV40/Zeo (InvitrogenCorp., Carlsbad, Calif.), which contains the ble gene fromStreptoalloteichus hindustanus flanked by an SV40 promoter andterminator, was also digested with NcoI to yield a 3201-bp and a 314-bpfragment. The 3201-bp fragment was agarose gel-purified and ligated tothe 463-bp NcoI fragment from p7TUB to yield pTUBZEO-11 (SEQ ID NO:8),depicted in FIG. 1.

Next, plasmid pTUBZEO-11 was digested with SphI, and a resulting 1122-bpfragment that contained the ble gene flanked by the Schizochytriumα-tubulin promoter and the SV40 terminator was agarose gel purified andligated to plasmid pUC19 (Messing, J. 1983. Meth. Enzymol. 101:20) thathad been linearized by digestion with SphI. The resulting plasmid wasnamed pTUBZEO11-2 (SEQ ID NO:9) and is depicted in FIGS. 2 and 4.Plasmid pTUBZEO11-2 is also referred to as pMON50000. In SEQ ID NO:9,the Schizochytrium α-tubulin promoter is contained within nucleotides441-894; the ble gene coding region is contained within nucleotides895-1269; and the SV40 terminator is contained within nucleotides1270-1524.

Example 2

This example describes the production of recombinant plasmids pMON50200,pMON50201, pMON50202, and pMON50203.

The native acetolactate synthase-encoding gene (als) from Schizochytriumsp. was isolated in the following manner. A cDNA clone(LIB81-028-Q1-E1-D9) was isolated from a Schizochytrium cDNA library andpartially sequenced (SEQ ID NO:11) as part of a large-scaleSchizochytrium sp. cDNA sequencing project. The nucleotide sequence wasdetermined by BLASTX homology to encode acetolactate synthase; e.g., theamino acid sequence deduced from bases 154 through 378 was 68% identicalwith amino acids 313 through 387 of ALS from Schizosaccharomyces pombe(GenBank Accession No. P36620). The full-length sequence of this clonedcDNA was then obtained, which indicated that the cDNA clone did notcontain the entire als coding region. In order to obtain the full-lengthals gene, a Schizochytrium genomic lambda library was probed usingstandard protocols (see e.g. Sambrook et. al., Molecular Cloning: ALaboratory Manual. Cold Spring Harbor Laboratory Press, 1989) with a372-bp digoxygenin (DIG)-labeled DNA probe (referred to as the ALS2probe). The ALS2 probe was generated via PCR using a nucleotide mix thatincluded DIG-11-UTP (Boehringer Mannheim Biochemicals GmbH, Germany),using forward primer PGR38 (SEQ ID NO:12) and reverse primer PGR39 (SEQID NO:13), which were based upon the sequence of cDNA cloneLIB81-028-Q1-E1-D9. One of the genomic clones identified with the ALS2probe, designated ALS-4A, was isolated and further characterized bySouthern hybridization blots using the DIG-labeled ALS2 probe. A 4.9-kbpfragment from AhdI-digested ALS-4A lambda DNA was found to hybridize tothe ALS2 probe. This fragment was isolated by agarose gel purification,treated with T4 DNA polymerase in order to generate blunt ends, and thenligated into SmaI-digested pBluescriptII KS+ (Stratagene Corp., LaJolla, Calif.) to form plasmid pMON50200 (depicted in FIG. 3-A). Thesequence of pMON50200 is given as SEQ ID NO:14. The sequence of theacetolactate synthase enzyme encoded by the Schizochytrium als gene isgiven as SEQ ID NO:15.

Plasmids pMON50201, pMON50202, and pMON50203 (depicted in FIGS. 3-B,3-C, and 3-D, respectively) were produced from plasmid pMON50200 bysite-directed mutagenesis such that the encoded acetolactate synthaseenzymes are no longer inhibited by certain compounds, includingsulfometuron methyl (SMM). These plasmids were constructed as follows.The “Transformerä” site-directed mutagenesis kit (Clontech Laboratories,Inc., Palo Alto, Calif.) was used to introduce the following mutationsinto plasmid pMON502000 according to the manufacturer's instructions. Anoligonucleotide selection primer, DM19 (SEQ ID NO:16), was used in allthree constructions; this primer leads to the conversion of a uniqueEcoRV site in the multiple cloning site of pMON50200 to an AatII site.Primer DM14 (SEQ ID NO:17) was used to change amino acid residue number595 in the encoded ALS enzyme from tryptophan to valine, while at thesame time introducing an AclI site in the gene sequence; the resultingplasmid is referred to as pMON50201 (SEQ ID NO:18). Likewise, primerDM15 (SEQ ID NO:20) was used to change amino acid residue number 192 inthe encoded ALS enzyme from a proline to a glutamine and to introduce aBsgI site in the als gene, resulting in plasmid pMON50202 (SEQ IDNO:21). To construct plasmid pMON50203 (SEQ ID NO:23), both DM14 andDM15 primers were used, resulting in an encoded ALS enzyme containingboth of the amino acid residue replacements described above. Thesequences of the mutant acetolactate synthase enzymes encoded byplasmids pMON50201, pMON50202, and pMON50203 are given as SEQ ID NO:19,SEQ ID NO:22, and SEQ ID NO:24, respectively.

Example 3

This example describes the genetic transformation of Schizochytrium sp.with the recombinant molecules described in Examples 1 and 2.

The strain used in this example in Schizochytrium sp. N230D, aderivative of American Type Culture Collection strain 20888 (ATCC,Manassas, Va.). For liquid cultures, cells were grown axenically inM50-3 medium at 30° C. with shaking at 200-300 rpm. M50-3 mediumcontains the following components: NaCl, 12.5 g; MgSO₄.7H₂O, 2.5 g; KCl,0.5 g; CaCl₂, 0.05 g; glucose, 30 g; Na-glutamate, 3 g; KH₂PO₄, 0.4 g;yeast extract, 1 g; NaHCO₃, 0.4 g; Na₂EDTA, 30 mg; FeCl₃.6H₂O, 1.2 mg;H₃BO₃, 34.2 mg; ZnSO₄.7H₂O; 0.67 mg; CoCl₂.6H₂O, 0.13 mg; NaMoO₄.2H₂O,25 μg; CuSO₄.5H₂O, 10 μg; NiSO₄.6H₂O, 0.26 mg; thiamine.HCl, 100 μg;biotin, 0.5 μg; cyanocobalamin, 0.5 μg, and deionized water (to oneliter); final pH adjusted to 7.0. For growth on solid media, cells weregrown at 30° C. on M50-3 medium or M1E-3 medium solidified by theaddition of 1.5% (w/v) agar. M1E-3 medium contains the followingcomponents: glucose, 4 g; (NH₄)₂SO₄, 0.75 g; Na₂SO₄, 5 g; MgSO₄.7H₂O, 2g; KH₂PO₄, 0.5 g; KCl, 0.5 g; CaCl₂.2H₂O, 0.1 g; MOPS buffer, 20.9 g;FeSO₄.4H₂O, 0.3 mg; MnCl₂.4H₂O, 0.1 mg; ZnSO₄.7H₂O; 80 μg; CoCl₂.6H₂O, 2μg; NaMoO₄.2H₂O, 1 μg; CuSO₄.5H₂O, 60 μg; NiSO₄.6H₂O, 80 μg;thiamine.HCl, 320 μg; CA-pantothenate, 320 μg; cyanocobalamin, 8 μg, anddeionized water (to one liter); final pH adjusted to 7.0.

The sensitivity of Schizochytrium sp. to Zeocin™ and SMM was determinedby including these inhibitors in solidified M1E-3 medium at variousconcentrations and spreading cells on the plates at densities similar tothose that are present during procedures used for selection ofrecombinant cells.

Genetic transformation of Schizochytrium cells was performed by particlebombardment (Sanford, J. C., F. D. Smith, and J. A. Russell. 1993. Meth.Enzymol. 217:483-509) using a Bio-Rad Biolistic PDS-1000/He ParticleDelivery System (Bio-Rad Laboratories, Hercules, Calif.). Schizochytriumsp. N230D cells were grown in liquid M50-3 medium to an optical densityat 680 nm (OD₆₈₀) of 0.4-0.8 (optical path length of 10 mm). An aliquotof cells corresponding to 1.0 OD₆₈₀ was briefly centrifuged, thesupernatant solution was removed, and the pelleted cells wereresuspended in 100 μl of sterile water. The resuspended cells were thenspread in a 4 to 6 cm circle onto a Petri plate containingagar-solidified medium (e.g., M50-3 or M1E-3 medium) and allowed to sitfor 30 to 60 min so that the excess water could be absorbed into thesolid medium; this is referred to as the target plate.

A 1.5 mg aliquot of gold microcarriers (0.6μ nominal diameter, availablefrom Bio-Rad Laboratories, Inc., Hercules, Calif.) was coated with 2.5μg of transformation plasmid DNA (i.e., plasmid pTUBZEO11-2, pMON50201,pMON50202, or pMON50203) as per the manufacturer's instructions(Biolistic® PDS-1000/He Particle Delivery System Instruction Manual;Bio-Rad Laboratories, Hercules, Calif.). The cells were bombarded withthe DNA-coated gold microcarriers using the following conditions: 1100psi burst disk, chamber vacuum of 25″ Hg, microcarrier launch assemblyplaced on the top shelf and the target plates placed on the middleshelf, giving a burst disk-to-stopping screen distance of 1.5-2 cm and astopping screen-to-target distance of approximately 7 cm. Afterbombardment, the cells were allowed to recover on the target plates for4-6 hours at 30° C. The cells were then rinsed off the target plateswith 1.5 ml sterile water, collected in a microfuge tube, centrifugedbriefly, and resuspended in 400 μl sterile water. One hundredmicroliters of the suspension were spread onto each of four M1E-3 platescontaining either 150-200 μg/ml Zeocin™ (Invitrogen Corp., Carlsbad,Calif.) or 25 μg/mL SMM. Zeocin™-containing plates were used to selectfor cells that had been transformed with plasmid pTUBZEO11-2, whereasSMM-containing plates were used to select for cells that had beentransformed with plasmids pMON50201, pMON50202, or pMON50203. The plateswere then incubated for 7-10 days at 30° C. Colonies that appeared to beresistant to the selective agent were then patched onto fresh M1E-3plates containing the same selective agent to confirm resistance. Thisprotocol typically results in the generation of 100-1000Zeocin™-resistant or SMM-resistant strains per bombardment.

Example 4

The following example demonstrates PCR analysis of transformedSchizochytrium cells.

PCR was used to confirm the presence of plasmid sequences in theputatively transformed strains that were resistant to the selectiveagents Zeocin™ or SMM. Template DNA from putative transformants andnon-recombinant Schizochytrium N230D cells was obtained by using asingle-use, plastic 1 μl inoculation loop to remove a small quantity ofcells (1-2 mm³) from resistant colonies that been patched onto agarplates (as described in Example 3). The cells were then resuspended in15-20 μl of 1% Triton X-100 in a microfuge tube, placed in a boilingwater bath for 10 minutes, and then centrifuged for 5 minutes at14,000×g. Portions of these extracts (1-3 μL) were used to provide thetemplate DNA for 25 μL PCR reactions using Taq DNA polymerase. To detectthe presence of pTUBZEO11-2 sequences in the Schizochytrium DNA, primersDM20 (SEQ ID NO:25) and DM21 (SEQ ID NO:26) were used; these primersanneal to the ble gene in plasmid pTUBZEO11-2 and amplify a 346-bp DNAfragment. The thermal profile used was as follows: 94° C. for 4 min;(94° C. for 45 sec, 52° C. for 45 sec, 72° C. for 2 min)×30; 72° C. for7 min. To detect the presence of pMON50201, pMON50202, or pMON50203sequences in the Schizochytrium DNA, primers BLA1 (SEQ ID NO:27) andBLA2 (SEQ ID NO:28) were used; these primers anneal to the bla(ampicillin-resistance) gene found in the vector backbone and amplify a1229-bp DNA fragment. The thermal profile used was as follows: 94° C.for 4 min; (94° C. for 45 sec, 55° C. for 45 sec, 72° C. for 2 min)×30;72° C. for 7 min. PCR products were analyzed by standard agarose gelelectrophoresis, followed by staining with ethidium bromide.

The results of these analyses confirm that the vast majority of strainsselected under these conditions are true transformants that containplasmid DNA. No PCR products of the correct size were generated whenusing template DNA from control Schizochytrium sp. N230D cells that hadnot been bombarded with the transformation plasmids.

Example 5

The following example describes Southern blot analyses of transformedSchizochytrium cells.

Southern hybridization blots were conducted using DNA isolated fromparental Schizochytrium N230D cells and several putative transformantsin order to confirm the presence of transformation vector DNA sequenceswithin the transformed cells. Southern blotting was conducted usingtechniques known to those skilled in the art (see e.g. Sambrook et. al.,Molecular Cloning: A Laboratory Manual. Cold Spring Harbor LaboratoryPress. 1989). DNA was isolated by the use of a “QIAamp” DNA purificationkit (Qiagen Inc., Valencia, Calif.), digested with various restrictionenzymes, separated by electrophoresis through agarose gels (0.8%-1.2%w/v), and then transferred to nylon membranes by alkaline capillarytransfer.

Detection of vector DNA in cells transformed with pTUBZEO11-2 wascarried out by use of the “Genius” DIG-based system (Boehringer MannheimBiochemicals GmbH, Germany), using as a hybridization probe a 346-bpDIG-labeled ble gene fragment generated via PCR with primers DM20 (SEQID NO:25) and DM21 (SEQ ID NO:26) and a nucleotide mix that includedDIG-11-UTP. Pre-hybridization of the membrane was carried out at 68° C.for 1 h in the hybridization buffer supplied in the Genius kit.Hybridization was carried out at 68° C. for 18 h in hybridization buffercontaining the ble gene probe that had been heat-denatured for 5 min at94° C. The membranes were then washed twice for 5 min with 50 mL2×SSC/0.1% SDS and twice for 15 min in 50 mL 0.1×SSC/0.1% SDS.Chemiluminescent detection of hybridizing DNA was performed as describedin the Genius kit instructions.

DNA from non-transformed Schizochytrium N230D cells did not hybridize tothe ble gene probe. Conversely, DNA from transformed cells did hybridizeto the probe as follows:

SphI:

SphI-digested DNA from transformed Schizochytrium cells contained a˜1100-bp DNA fragment that hybridized to the ble gene probe; thisfragment, which is also observed in SphI-digested pTUBZEO11-2 DNA,represents the entire ble gene expression cassette (including thetubulin gene promoter and SV40 terminator).

XhoI:

For each of the transformants tested, XhoI digestion of DNA resulted inhybridizing fragments larger than 15-20 kbp. XhoI does not cut withinpTUBZEO11-2, and therefore these results indicate that pTUBZEO11-2 doesnot appear to exist as an extrachromosomal element in transformed cells,but rather becomes integrated into the Schizochytrium chromosome.

NcoI or HindIII:

These enzymes both cut once within pTUBZEO11-2. Digestion oftransformant DNA with either of these enzymes typically led to aprominent hybridizing fragment that comigrated with linearizedpTUBZEO11-2 vector (i.e., ˜3.8 kbp). This suggests that the vector canintegrate in the chromosome in the form of tandem repeats.

Example 6

This example demonstrates homologous recombination in Schizochytrium.

The following experiments were conducted to demonstrate that homologousrecombination can occur in Schizochytrium between endogenous native DNAsequences and homologous DNA sequences present in recombinant DNAmolecules introduced into the cells. This type of homologousrecombination can be very beneficial for producing recombinant strainswith desirable properties. For example, homologous recombination can beused to inactivate endogenous genes by the targeted insertion of foreigngenetic sequences. Additionally, homologous recombination can be used toreplace an endogenous gene or portion thereof with an altered form ofthe gene such that the recombinant cells exhibit novel properties.

Homologous recombination was shown to occur in Schizochytrium cellstransformed with plasmid pMON50202, which contains a mutation in theSchizochytrium als gene. This mutation introduces a BsgI site at byposition 571 of the als coding region. There is a naturally occurringBsgI site at by position 1324 of the als coding region. Therefore,Southern blots of BsgI-digested Schizochytrium DNA can be used todiscern the native als gene from the recombinant mutant als gene. Forthese experiments, an als-specific hybridization probe was produced viaPCR using a nucleotide mix that included DIG-11-UTP (Boehringer MannheimBiochemicals GmbH, Germany), forward primer PGR28 (SEQ ID NO:32),reverse primer PGR30 (SEQ ID NO:33), and a small amount of pMON50200 asthe template. The resulting 323-bp DIG-labeled hybridization probe wasreferred to as ALS1.

DNA from non-recombinant Schizochytrium N230D cells was digested withBsgI and AhdI separately, subjected to agarose gel electrophoresis,transferred to a nylon membrane, and then probed with the ALS1 probeusing procedures essentially the same as those described in Example 5.The ALS1 probe labeled a 1.76-kbp fragment of BsgI-digested DNA and a4.9-kbp fragment of AhdI-digested DNA.

Southern blots of BsgI- and AhdI-digested DNA from various recombinantstrains that had been transformed with pMON50202 were also probed withthe ALS 1 probe. In some cases, the 1.76-kbp BsgI fragment was notpresent, and instead a 0.75-kbp fragment was labeled, corresponding tothe 753-bp BsgI fragment present in pMON50202. A 4.9-kbp AhdI fragmentwas labeled in these recombinant strains, however, indicating that therecombinant, mutant als gene had recombined with the native als gene viadouble-crossover homologous recombination.

Single-crossover homologous recombination was also observed to occur inrecombinant strains transformed with pMON50202. In these cases, both the1.76-kbp and 0.75-kbp BsgI fragments were labeled in the Southern blotsof DNA from the recombinant strains, but the 4.9-kbp AhdI fragment wasreplaced by larger labeled fragments, indicating that the entirepMON50202 vector had inserted into the native als gene, either as asingle copy or as tandem repeats.

Additional evidence for homologous recombination in Schizochytrium wasobtained by the introduction of recombinant DNA molecules containing atruncated, mutant als gene such that the incomplete ALS enzyme encodedby the truncated gene was nonfunctional. This truncated gene wasproduced by digesting pMON50202 with ClaI and HindIII to yield a 2.8-kbpfragment, thereby removing the last 388 bp of the als coding sequencealong with the als terminator region. This 2.8-kbp fragment was ligatedinto pBluescriptII KS+ (Stratagene Corp., La Jolla, Calif.) that hadbeen digested with ClaI and HindIII, yielding plasmid pAR2. Plasmid pAR2would only be expected to confer resistance to SMM in transformedSchizochytrium cells if a functional, mutant als gene was restored intransformed strains via homologous recombination between the native alsgene and the truncated mutant als gene present in pAR2. This constructwas introduced into Schizochytrium N230D cells by particle bombardment,and SHIM-resistant strains were isolated as described in Example 3.Southern blot analysis of BsgI-digested DNA from the transformants,carried out as described earlier in this example, indicated thathomologous recombination had clearly occurred in these strains; i.e., a1.76-kbp BsgI fragment hybridized to the ALS1 probe in non-recombinantcells, but this was replaced by a 0.75-kbp hybridizing fragment in cellsthat had been transformed with pAR2.

Example 7

This example describes the use of transformation vector pTUBZEO11-2 orpMON50202 to produce via co-transformation strains that containadditional foreign DNA molecules that are not linked to a selectablemarker gene.

Co-transformation was achieved by simultaneous introduction ofpTUBZEO11-2 and an additional plasmid containing any of several genes.The plasmids were co-precipitated on the gold particles as described inExample 3, using 2.5 μg of each plasmid. After the bombardment of targetcells with the plasmid-coated gold particles, recombinant strains wereselected on Zeocin™-containing agar plates as described in Example 1.The presence of the second, non-selected plasmid was then confirmed byPCR analysis or by Southern blot hybridization. Very highco-transformation frequencies (e.g., 50-90%) were typically achieved.For example, the plasmid pTR202, which contains the Caenorhabdituselegans fat-1 gene (Spychalla et al., 1997. Proc. Natl. Acad. Sci.U.S.A. 94, 1142-1147) linked to the Schizochytrium tubulin gene promoterand terminator, was introduced via the method provided in this example,and about 68% of the resulting Zeocin™-resistant strains were shown byPCR to contain the fat-1 gene (See Table 1). Similar results areobserved when pMON50202 and an additional plasmid are co-introduced,followed by selection of transformed cells on SMM-containing solidmedium. This co-transformation method can be used to introduce anyforeign DNA desired.

TABLE 1 Efficiencies of co-transformation using the selectable markerplasmid pTubZeo11-2 and plasmids containing various fad genes.Zeocin^(R)-resistant transformants were screened for fad DNA sequencesvia PCR. Introduced fad #containing fad gene Co-transformation Gene #Zeocin^(R) strains tested efficiency syn_fat1 17/25 68% nat_fat1 24/2596% mut_fat1 21/25 84% desB 20/25 80%

The transformation systems described in these examples represent asignificant advance in the ability to genetically manipulateSchizochytrium, which is the most productive organism known for thefermentative production of lipid-based compounds. The availability oftwo independent transformation systems, along with the highco-transformation efficiencies that occur, should allow stacking ofmultiple traits in engineered strains. Furthermore, the apparentpresence of homologous recombination in this microalga should allow thedevelopment of gene knockout procedures in order to identify thefunctions of unknown genes and to eliminate undesirable traits inproduction strains. The present inventors are currently using thesesystems to alter fatty acid metabolism in Schizochytrium, and areexploring possibilities for using this species and related microalgae(e.g., Thraustochytrium) for the production of carotenoids, sterols, andother lipoidal compounds.

While various embodiments of the present invention have been describedin detail, it is apparent that modifications and adaptations of thoseembodiments will occur to those skilled in the art. It is to beexpressly understood, however, that such modifications and adaptationsare within the scope of the present invention, as set forth in thefollowing claims.

The invention claimed is:
 1. A method for production of a protein,comprising: culturing a recombinant microorganism of the OrderThraustochytriales in a medium, wherein the recombinant microorganismcomprises an isolated nucleic acid molecule comprising a foreign genethat encodes the protein, to produce the protein; and recovering theprotein.
 2. The method of claim 1, wherein the isolated nucleic acidmolecule further comprises one or more transcription control sequencesand the foreign gene is operatively linked to the one or moretranscription control sequences.
 3. The method of claim 2, wherein theone or more transcription control sequences comprise aThraustochytriales transcription control sequence.
 4. The method ofclaim 3, wherein the Thraustochytriales transcription control sequenceis selected from the group consisting of Thraustochytriales promotersequences, Thraustochytriales enhancer sequences, Thraustochytrialesoperator sequences, Thraustochytriales repressor sequences, andcombinations thereof.
 5. The method of claim 4, wherein at least oneThraustochytriales transcription control sequence is aThraustochytriales promoter sequence.
 6. The method of claim 5, whereinthe Thraustochytriales promoter sequence is selected from the groupconsisting of a Thraustochytriales acetolactate synthase promotersequence, Thraustochytriales α-tubulin promoter sequence,Thraustochytriales polyketide synthase system promoter sequence, andThraustochytriales fatty acid desaturase promoter sequence.
 7. Themethod of claim 1, wherein the isolated nucleic acid molecule furthercomprises a signal segment nucleic acid sequence.
 8. The method of claim1, wherein the protein is secreted into the medium, and wherein theprotein is recovered from the medium.
 9. The method of claim 1, whereinthe microorganism is from a genus selected from the group consisting ofThraustochytrium, Labyrinthuloides, Japonochytrium, and Schizochytrium.10. The method of claim 9, wherein the microorganism is from a speciesselected from the group consisting of Schizochytrium sp., Schizochytriumaggregatum, Schizochytrium limacinum, Schizochytrium minutum,Thraustochytrium sp., Thraustochytrium striatum, Thraustochytriumaureum, Thraustochytrium roseum, and Japonochytrium sp.
 11. A method forproduction of a protein, comprising: culturing a recombinantmicroorganism of the Order Thraustochytriales in a medium, wherein therecombinant microorganism comprises an isolated nucleic acid moleculecomprising a foreign gene that encodes the protein, to produce theprotein; and harvesting the microorganism comprising the protein. 12.The method of claim 11, wherein the microorganism is harvested as abiomass.